Treatment of α-galactosidase A deficiency

ABSTRACT

The invention provides methods of treating α-galactosidase A deficiency. Dosage forms, methods of administration, and methods of analyzing human α-galactosidase A are also included.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/403,618, filed Apr. 13, 2006, now U.S. Pat. No. 7,833,742, which is adivisional of U.S. application Ser. No. 10/423,225, filed Apr. 25, 2003,now abandoned, which claims priority to U.S. Provisional ApplicationSer. No. 60/375,584, filed Apr. 25, 2002, the contents of all of whichare incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

This invention relates to improved α-galactosidase A compositions forthe treatment of α-galactosidase A deficiencies including Fabry disease.

BACKGROUND OF THE INVENTION

Fabry disease is an X-linked inherited lysosomal storage diseasecharacterized by severe renal impairment; angiokeratomas, and/orcardiovascular abnormalities, including ventricular enlargement andmitral valve insufficiency. Fabry disease also affects the peripheralnervous system, causing episodes of agonizing, burning pain in theextremities.

Fabry disease is caused by a deficiency in the enzyme α-galactosidase A(α-Gal A). The pathophysiology of Fabry Disease is well established: dueto a lack of the lysosomal enzyme α-galactosidase A (α-Gal A), there isaccumulation of globotriaosylceramide (Gb₃) throughout the body.

Due to the X-linked inheritance pattern of the disease, the majority ofFabry disease patients are male. Severely affected female heterozygotesare often observed, though female heterozygotes may become symptomaticlater in life. A variant of Fabry disease correlates with leftventricular hypertrophy and cardiac disease. Nakano et al., New Engl. JMed. 333: 288-293 (1995). The cDNA and gene encoding human α-Gal A havebeen isolated and sequenced. Human α-Gal A is expressed as a 429-aminoacid polypeptide, of which the N-terminal 31 amino acids are the signalpeptide. The human enzyme has been expressed in Chinese Hamster Ovary(CHO) cells (Desnick et al., U.S. Pat. No. 5,356,804; Ioannou et al., JCell Biol. 119: 1137 (1992)); insect cells (Calhoun et al., WO90/11353); and human cells (Selden et al., U.S. Pat. Nos. 6,083,725 and6,458,574B1). Enzyme replacement therapy is a currently used method oftreatment for Fabry disease.

SUMMARY OF THE INVENTION

By understanding the pharmacokinetics and modification profile (e.g.,carbohydrate, phosphate or sialylation modification) of human α-Gal A,we have developed novel pharmaceutical compositions of α-Gal A, kits fortreatment of α-Gal A deficiency, methods of selecting an appropriatedose of α-Gal A for a patient, and methods of treating α-Gal Adeficiency using such compositions. Also provided are methods ofevaluating α-Gal A preparations, samples, batches, and the like, e.g.,methods of quality control and determination of bioequivalence, e.g.,with reference to the α-Gal A compositions described herein.

The α-Gal A dosing and administration strategies described herein reducethe amount and cost of α-Gal A required for α-Gal A replacement therapyand also reduce the required number of dose administrations.

Accordingly, in one aspect, the invention features a pharmaceuticalcomposition that includes a human α-Galactosidase A (α-Gal A))preparation. At doses below serum or plasma clearance saturation levels,serum clearance of the α-Gal A preparation from the circulation ispreferably less than 4 mL/min/kg on the linear portion of the AUC vs.dose curve, more preferably less than about 3.5, 3, or 2.5 mL/min/kg, onthe linear portion of the AUC vs. dose curve. The α-Gal A preparationcan have an exponent “b” for the allometric scaling equation forclearance from circulation (serum or plasma) in mammals, Y=a (BW)^(b),of at least 0.85, where Y is the clearance rate of α-Gal A (ml/min), “a”is a non-specific constant, and BW is body weight. The exponent “b” ispreferably at least 0.88, more preferably at least 0.90, and mostpreferably at least 0.92 or at least 0.94.

In one embodiment, the α-Gal A is produced from human cells, e.g.,primary human cells, e.g., primary human fibroblasts or a continuoushuman cell line. The cells and/or the α-Gal A preparation isolated fromthe cells can be modified to provide an α-Gal A preparation withdesirable glycosylation, phosphorylation or sialylation characteristics.

In another embodiment, the α-Gal A is produced from non-human cells,e.g., CHO cells. The cells and/or the α-Gal A preparation isolated fromthe cells can be modified to provide an α-Gal A preparation withdesirable glycosylation, phosphorylation or sialylation characteristics.

In another aspect, the invention features a kit for the treatment ofα-Gal A deficiency. The kit includes (a) a human α-Gal A glycoproteinpreparation, where at doses below serum or plasma clearance saturationlevels, serum clearance of the α-Gal A preparation from the circulationis preferably less than 4 mL/min/kg on the linear portion of thearea-under-the-curve (AUC) vs. dose curve, more preferably less thanabout 3.5, 3, or 2.5 mL/min/Kg, on the linear portion of the AUC vs.dose curve, and (b) instructions to administer the preparation to asubject in need thereof.

The kit can also include instructions to administer a unit dose of theα-Gal A preparation of between about 0.05 mg and 2.0 mg per kilogram ofbody weight of the subject (mg/kg). In some embodiments, the kitincludes instructions to administer a unit dose of the α-Gal Apreparation of between 0.05 and 2.0 mg/kg, preferably between about 0.05and 1.0 mg/kg, more preferably between about 0.05 and 0.5 mg/kg, e.g.,and between 0.05 and less than 0.3 mg/kg. In one embodiment, the unitdose is less than 0.3 mg/kg. For example, the kit can includeinstructions to administer a unit dose of the α-Gal A preparation ofabout 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 mg per kilogramof body weight.

In other embodiments, the kit includes instructions to administer a unitdose of the α-Gal A preparation of between about 0.1×10⁶ U/kg and 10×10⁶U/kg. In some embodiments, the kit includes instructions to administer aunit dose of the α-Gal A preparation of between 0.1×10⁶ U/kg and 5×10⁶U/kg, preferably between about 0.1×10⁶ U/kg and 3×10⁶ U/kg. For example,the kit can include instructions to administer a unit dose of the α-GalA preparation of about 0.1, 0.2, 0.3, 0.5, 1, 2, 3, 5 or up to 10×10⁶U/kg.

In some embodiments, the kit also includes instructions to administerthe unit dose no more than once every 7 days. For example, theinstructions can include instructions to administer the unit dose nomore than once every 7 days, 10 days, 14 days, 21 days, 4 weeks, 6weeks, 8 weeks or 10 weeks.

In another aspect, the invention features a kit for the treatment ofα-Gal A deficiency. The kit includes a human α-Gal A glycoproteinpreparation and one or more of the following instructions: (a)instructions to administer the preparation to a subject in need thereofat a unit dose of between 0.05 and 2.0 mg/kg, preferably between about0.05 and 1.0 mg/kg, more preferably between about 0.05 and 0.5 mg/kg,e.g., between 0.05 and less than 0.3 mg/kg; (b) instructions toadminister a unit dose of the α-Gal A preparation of between 0.1×10⁶U/kg and 10×10⁶ U/kg, e.g., between 0.1×10⁶ U/kg and 5×10⁶ U/kg,preferably between about 0.1×10⁶ U/kg and 3×10⁶ U/kg; or (c)instructions to administer the preparation no more than about once every8 weeks, 6 weeks, 4 weeks, 2.1 days, 14 days, 10 days, or 7 days.

The reagents of a kit described herein may be packaged in containers inpredetermined amounts. A kit embodying features of the presentinvention, generally designated by the numeral 2, is illustrated in FIG.16. Kit 2 is comprised of the following major elements: packaging 4, anα-Gal A preparation described herein 6, and instructions 8. Optionally,the kit may include an additional agent 10. The additional agent can be,e.g., a pharmaceutical buffer or solution, e.g., for dissolving ordiluting the α-Gal A preparation 6. Instructions 8 can be, e.g., printedmaterial on how to administer the preparation 6 and may includeinformation on suitable dosage. Preferred instructions compriseinstructions to administer the α-Gal A preparation 6 in a unit dosedescribed herein. Packaging 4 is a box-like structure for holding a vial(or number of vials) containing an α-Gal A preparation of the invention6, instructions 8, and, optionally, a vial (or number of vials)containing an agent 10. An individual skilled in the art can readilymodify packaging 4 to suit individual needs.

The invention also features a method of selecting a unit dose range ofα-Gal A for treatment of a subject having an α-Gal A deficiency. Themethod includes: providing the body weight of a subject, e.g., weighingthe subject or obtaining the subject's body weight from the subject,from a health care provider of the subject, or from a database; anddetermining the value of the range between 0.05 mg and 2 mg (e.g.,between 0.05 and 0.5 mg or between 0.05 and less than 0.3 mg) of α-Gal Aper kilogram of body weight of the subject. The selected unit dose rangecan be used to select a regimen of α-Gal A replacement therapy for thesubject. The method can also include evaluating the subject for one ormore of: basal α-Gal A levels, e.g., α-Gal A serum concentration;cardiovascular function; renal function; liver function, age, sex.

In a preferred embodiment, the unit dose saturates liver uptake of theα-Gal A by having C_(max) (maximum serum concentration following druginfusion) greater than 2×10⁻⁹ M.

In another aspect, the invention also features a method of treating asubject having or at risk for having α-Gal A deficiency. The methodincludes administering to a subject in need thereof a human α-Gal Aglycoprotein preparation, where at doses below serum or plasma clearancesaturation levels, serum clearance of the α-Gal A preparation followingintravenous infusion from the circulation is preferably less than 4mL/min/kg on the linear portion of the AUC vs. dose curve, morepreferably less than about 3.5, 3, or 2.5 mL/min/kg, on the linearportion of the AUC vs. dose curve, e.g., a human α-Gal A glycoprotein,preparation described herein above. As described elsewhere herein theunit dose administered preferably saturates liver uptake of the α-Gal A.

In a preferred embodiment, the preparation is administeredintravenously, although it may be formulated for oral, subcutaneous, orintrathecal, administration, as described elsewhere herein.

In another aspect, the invention includes a method of treating a subjecthaving or at risk for α-Gal A deficiency. The method includes one ormore of (a)-(c): (a) administering to a subject in need thereof a humanα-Gal A glycoprotein preparation at a unit dose of between about 0.05and 2.0 mg per kilogram of body weight, preferably between 0.05 and 1.0mg/kg or between 0.05 and 0.5 mg/kg, e.g., between 0.05 and less than0.3 mg/kg, e.g., about 0.25, 0.20, 0.15 or 0.1 mg per kilogram of bodyweight of the subject; (b) administering to a subject in need thereof ahuman α-Gal A glycoprotein preparation at a unit dose of the α-Gal Apreparation of between 0.1×10⁶ U/kg and 10×10⁶ U/kg, e.g., between0.1×10⁶ U/kg and 5×10⁶ U/kg, preferably between about 0.1×10⁶ U/kg and3×10⁶ U/kg; (c) administering to a subject in need thereof a human α-GalA glycoprotein preparation no more than once, every 7 days, e.g., nomore than once every 10 days, 14 days, 21 days, 4 weeks, 6 weeks, or 8weeks. In some embodiments, there are at least 7, 10, 14, 21, 30 or 60days between each administration. In some embodiments, the preparationis administered over a period of at least 8, 16, 24, 36; 48, weeks oreven longer, e.g., at least 1, 2, or 3 years.

In one embodiment, the human α-Gal A glycoprotein preparation isadministered at least twice, preferably 3, 4, 5, 6 times or more, but nomore than once every 7 days, preferably 10 days, more preferably 14 daysor more, e.g., 21 days, 4 weeks, 6 weeks, 7 weeks, 8 weeks or more.

In a preferred embodiment, the unit dose saturates liver uptake of theα-Gal A, so as to allow administered α-Gal A to bypass the liver and beavailable to other tissues in the body.

In a preferred embodiment, the preparation is administeredintravenously.

In another aspect, the invention features a unit dose of human α-Gal Adescribed herein packaged in a container, e.g., a glass or plasticcontainer or delivery device, e.g., a syringe. The unit dose isequivalent to between 0.05 and 0.2 mg/kg, e.g., between 0.05 and 1.0mg/kg, preferably between 0.05 and 0.5 mg/kg, more preferably between0.05 and less than 0.3 mg/kg of body weight of the subject for which itis intended. The activity of the α-Gal A preparation is generallybetween about 2.0 and 4.5×10⁶ U/mg. For example, the container ordelivery device can include between 2.0 and 32.0 mg of human α-Gal Adescribed herein for an adult unit dose, e.g., the container or deliverydevice can include about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25 or 30 mgof α-Gal A for an adult dose.

Although not bound by any theory, it is believed that the α-Gal Apreparations described herein can be predominantly cleared from theblood through mannose-6-phosphate (M6P) receptors. In preferredembodiments, less than 25%, 20%, 16%, 14% (as measured between 40 hoursand 50 hours, e.g., approximately 44 hours, after dosing) or less of theα-Gal A preparation, e.g., a preparation described herein, is taken intothe liver upon administration to a subject. At doses below serum orplasma clearance saturation levels, serum clearance of the α-Gal Apreparation from the circulation is preferably less than 4 mL/min/kg onthe linear portion of the AUC vs. dose curve, more preferably less thanabout 3.5, 3, or 2.5 mL/min/kg, on the linear portion of the AUC vs.dose curve. An α-Gal A preparation described herein exhibits a liversaturation curve as follows:mg α-Gal A/liver=2.1 mg(1−e ^(−Dose/4.7)),

where dose is the total dose (in mg) administered to a typical 75 kgpatient. The coefficient of variation (CV) can be, e.g., about 0.40.(Doses and amounts would be adjusted accordingly for larger or smallerpatients).

In some embodiments, the α-Gal A preparation of the compositions,methods and kits described herein is isolated from human cellsgenetically engineered to produce α-Gal A. In other embodiments, theα-Gal A preparation can be isolated from non-human cells (e.g., CHOcells), where the cell has been genetically engineered to produce α-GalA. In some embodiments, one or more of: the α-Gal A expressionconstruct, the human or non-human cells, or the α-Gal A isolated fromthe human or non-human cells can be modified to provide an α-Gal Apreparation with altered glycosylation, e.g., altered glycan,sialylation or phosphate structures. For example, a non-human cellgenetically engineered to produce a human α-Gal A (or the purified α-GalA) can be modified to mimic the glycosylation characteristics of α-Gal Aproduced in human cells. In one embodiment, the cells can be modified,e.g., genetically engineered, to express one or more exogenous α-Gal Amodifying enzyme, e.g., a glycosidase, glycosyl transferase, phosphoryltransferase, or sialyl transferase. In one embodiment, the α-Gal Acoding sequence can be modified to have more or fewer (preferably more)glycosylation sites. In another embodiment, the cells can be exposed toone or more inhibitor or other modulator of glycosylation enzymes, e.g.,kifunensine or swainsonine. In yet another embodiment, the α-Gal A, onceisolated from the cells, can be modified, e.g., cleaved or chemicallymodified (e.g. by changing the number of moles of sialic acid and/ormannose-6-phosphate per mole of α-Gal A), e.g., with a phosphataseinhibitor, kinase, glycosidase, glycosyl transferase, phosphoryltransferase, or sialyl transferase.

In preferred embodiments, an α-Gal A preparation described herein isenriched in neutral, mono-sialylated and di-sialylated glycan structures(combined) relative to more highly sialylated structures such astri-sialylated and tetra-sialylated structures. For example, a preferredα-Gal A preparation has one or more of: (a) at least about 22% neutralglycans, e.g., at least about 25% or 30% neutral glycans; (b) at leastabout 15%, 20%, or 25% mono-sialylated glycans; (c) at least about 35%,preferably at least about 40%, 45%, or 50% neutral and mono-sialylatedglycans combined; (d) at least about 75%, 76%, 78% or more neutral,mono- and di-sialylated glycans combined; and (e) less than about 35%,preferably less than about 25%, 20%, 18% or about 15% tri- andtetra-sialylated glycan structures combined.

In preferred embodiments, an α-Gal A preparation described herein has,on average, more than one complex glycan per monomer, preferably atleast 50% complex glycans per monomer, e.g., an average of 1.5 complexglycans or, more per monomer.

In preferred embodiments, an α-Gal A preparation described herein has atleast 5%, preferably at least 7%, 10% or 15% neutral glycans.

In preferred embodiments, an α-Gal A preparation described herein hasless than 45% phosphorylated glycans. For example, the preparation hasless than about 35%, 30%, 25%, or 20% phosphorylated glycans.

In preferred embodiments, an α-Gal A preparation described herein has atotal proportion of sialylated glycans greater than about 45%, e.g.,greater than 50% or 55%.

In a preferred embodiment, the ratio of sialic acid tomannose-6-phosphate in the α-Gal A preparation (on a mole per molebasis) is greater than 1.5 to 1, preferably greater than 2 to 1, morepreferably greater than 3 to 1, most preferably greater than 3.5 to 1 orhigher.

In one embodiment, the percent ratio of sialylated glycans tophosphorylated glycans is greater than 1, preferably greater than 1.5,more preferably greater than 2, e.g., greater than about 2.5 or 3.

The α-Gal A compositions and methods described herein are useful fortreatment of individuals with α-Gal A deficiency. The α-Gal Acompositions and methods described herein provide treatments that arecost effective and minimize the required dosage and frequency ofadministrations of α-Gal A.

In another aspect, the invention features various methods of evaluating,e.g., analyzing, selecting or classifying an α-Gal A preparation,sample, batch or other composition. The methods can be used to determinethe structural and/or biological parameters (e.g., the carbohydratecomposition, phosphate profile, sialylation profile, tissuedistribution, or serum clearance characteristics) of the preparation orsample. By way of a non-limiting example, the methods are used todetermine if the preparation or sample has one or more physical orfunctional property of an α-Gal A described herein. For example, one cancompare a sample α-Gal A composition to a reference α-Gal A composition,e.g., a human α-Gal A composition described herein, e.g., a human α-GalA having desirable pharmacokinetic or biological properties, such as ahuman α-Gal A prepared from human cells, e.g., human fibroblasts. Themethods are useful, inter alia, for quality control and/orbioequivalence studies of α-Gal A preparations.

In one aspect, the method includes obtaining or providing a test α-Gal Apreparation and determining if the preparation has at least one(preferably at least two, more preferably at least three or more, e.g.,at least four, five, six or seven) of the following structuralcharacteristics: (1) is enriched in neutral, mono-sialylated anddi-sialylated glycan structures (combined) relative to more highlysialylated structures, for example, has (i) at least about 22% neutralglycans, e.g., at least about 25% or 30% neutral glycans, (ii) at leastabout 15%, 20%, or 25% mono-sialylated glycans, (iii) at least about35%, preferably at least about 40%, 45%, or 50% neutral andmono-sialylated glycans combined, and/or (iv) at least about 75%, 76%,78% or more neutral, mono- and di-sialylated glycans combined; (2) hasless than about 35%, preferably less than about 25%, 20%, 18% or about15% tri- and tetra-sialylated glycan structures combined; (3) has atleast 50%, preferably at least 67% complex glycans; (4) has less thanabout 45% phosphorylated glycans, preferably less than about 35%, morepreferably less than about 30%, 25% or 20% phosphorylated glycans; (5)has greater than about 45%, preferably greater than about 50 or 55%sialylated glycans; (6) has a ratio of sialic acid tomannose-6-phosphate on a mole per mole basis greater than 1.5 to 1,preferably greater than about 2 to 1; more preferably greater than about3 to 1 or 3.5 to 1; and (7) has a ratio of sialylated glycans tophosphorylated glycans greater than 1, preferably greater than 1.5, morepreferably greater than about 2, 2.5 or 3; and/or has one or more of thefollowing biological or pharmacokinetic characteristics: (a) serumclearance from human circulation is less than 4 mL/min/kg on the linearportion of the AUC vs. dose curve, more preferably less than about 3.5,3, or 2.5 mL/min/kg, on the linear portion of the AUC vs. dose curve;(b) the preparation is preferentially targeted to capillary/vascularendothelial cells, renal glomerular epithelial cells (podocytes) andglomerular mesangial cells, renal endothelial cells, cardiac myocytes,liver endothelial cells, liver sinusoidal cells, pulmonary cells, and/orneural cells; and (c) is not taken up by liver hepatocytes.

A test preparation that has one or more of the aforementionedcharacteristics can be selected, classified, formulated, packaged, orpassed on to other downstream processing. For example, such apreparation can be selected for a particular pharmaceutical use. Thepossession by the test preparation of one or more (preferably at leasttwo, more preferably at least three or more, e.g., at least four, five,six or seven) of the aforementioned structural parameters (1)-(7) ispositively correlated with (and can thus be used to predict) desirablepharmacokinetic parameters or biological activity, e.g., one or more ofthe biological or pharmacokinetic characteristics (a-(c). Thecorrelation predictive information can be used, e.g., to design an α-GalA therapeutic preparation for a specific patient or a specific variantof Fabry disease (e.g., renal variant Fabry disease or cardiac variantFabry disease). The correlation or prediction information can berecorded (e.g., in a print or computer readable medium).

In some embodiments, a biological activity or pharmacokinetic parameterof the test α-Gal A preparation or sample is predicted from itscarbohydrate signature. In other embodiments, a biological activity orpharmacokinetic parameter of the preparation or sample is determinedexperimentally.

The result of the determination (which can be, e.g., a value for any of:the amount of neutral, mono-, di-, tri- or tetra-sialylated glycans orcombinations thereof; the amount of complex glycans; the amount ofphosphorylated glycans; the amount of sialylated glycans; the ratio ofsialic acid to mannose-6-phosphate on a mole per mole basis, or theratio of sialylated glycans to phosphorylated glycans), is preferablyentered into a record, e.g., a print or computer-readable record, suchas a laboratory record or dataset. The record can include otherinformation, such as a specific sample identifier for the preparation, adate, an operator of the method, or information about the enzymaticactivity, source, method of purification or biological activity of thepreparation. The record can be used to store or provide informationabout the test preparation. For example, the record can be used toprovide information (e.g., to the government, a health care provider,insurance company or patient) related to the α-Gal A preparation or itsuse e.g., in the form of informational, marketing or instructionalmaterial, e.g., print material or computer readable material (e.g., alabel). The record or information derived from the record can be used,e.g., to identify the test preparation as suitable or unsuitable forpharmaceutical or therapeutic use. For example, a test α-Gal Apreparation determined to have one or more of the aforementionedstructural parameters (1)-(7) can be identified as having desirablepharmacokinetic parameters or biological activity (e.g., theaforementioned parameters (a)-(c).

The methods described herein can also be used to compare batch-to-batchvariation of an α-Gal A preparation. In this case, any of the structuralor pharmacokinetic parameters described hereinabove can be evaluated fora plurality of α-Gal A batches, e.g., different batches made from thesame purification protocol. In a preferred embodiment, the methodincludes selecting a batch with less than a preselected range ofvariation (e.g., less than 10%, preferably less than 5%, more preferablyless than 2.5% or less variation) from one or more of the aforementionedstructural or biological parameters (1)-(7) or (a)-(c). When multiplepreparations are analyzed (e.g., different batches of an α-Gal Apreparation), entering the result of the determinations into a recordcan include generating a dataset of the determinations, e.g., a print orcomputer-readable dataset. The dataset can include a correlation of adetermined structural characteristic with a predicted or experimentallyevaluated biological activity or pharmacokinetic parameter.

The α-Gal A sample to be tested can be derived from any cell, butpreferably is derived from a mammalian cell, e.g., a human or non-humancell, such as a CHO cell. In some embodiments, the carbohydratesignature of the sample has been modified, e.g., by art-recognizedmethods, before the determination step is performed, e.g., byglycoengineering, e.g., as described herein, by treatment with aglycosidation enzyme such as a glycosyl transferase or glycosidase, ortreatment of the cell or preparation with a phosphoryl transferase,sialyl transferase, phosphatase inhibitor, kinase, or inhibitor ofglycosylation; or by co-expression in the cell (e.g., viaco-transfection) of a DNA encoding any of the foregoing enzymes or othercarbohydrate modifying enzymes.

The carbohydrate signature of the sample can be obtained by methodsknown in the art, e.g., by ion exchange chromatography, high performanceanion exchange (HPAE) chromatography, high performance liquidchromatography (HPLC), or mass spectroscopy. Evaluating the carbohydratesignature can include evaluating the composition, charge,phosphorylation, and/or sialylation of the glycans of the preparation.

In another aspect, the invention features a method of producing a humanα-Gal A preparation (e.g., an improved α-Gal A preparation). The methodincludes providing a human α-Gal A preparation harvested from a cell;and modifying the glycan structure of the α-Gal A preparation to matchone or more (preferably at least two, more preferably at least three ormore, e.g., at least four, five, six or seven) of the followingparameters: (1) enrichment in neutral, mono-sialylated and di-sialylatedglycan structures (combined) relative to more highly sialylatedstructures, for example, has (i) least about 22% neutral glycans, e.g.,at least about 25% or 30% neutral glycans, (ii) at least about 15%, 20%,or 25% mono-sialylated glycans, (iii) at least about 35%, preferably atleast about 40%, 45%, or 50% neutral and mono-sialylated glycanscombined, and/or (iv) at least about 75%, 76%, 78% or more neutral,mono- and di-sialylated glycans combined; (2) less than about 35%,preferably less than about 25%, 20%, 18% or about 15% tri- andtetra-sialylated glycan structures combined; (3) at least 50%,preferably at least 67% complex glycans; (4) less than about 45%phosphorylated glycans, preferably less than about 35%, more preferablyless than about 30%, 25% or 20% phosphorylated glycans; (5) greater thanabout 45%, preferably greater than about 50 or 55% sialylated glycans;(6) a ratio of sialic acid to mannose-6-phosphate on a mole per molebasis greater than 1.5 to 1, preferably greater than about 2 to 1, morepreferably greater than about 3 to 1 or 3.5 to 1; and (7) a ratio ofsialylated glycans to phosphorylated glycans greater than 1, preferablygreater than 1.5, more preferably greater than about 2, 2.5 or 3. Theglycan structure can be modified by methods known in the art, e.g., byglycoengineering (e.g., by genetically engineering the cell to produce ahuman α-Gal A having a non-naturally occurring glycosylation site;and/or genetically engineering the cell to produce a glucosidase,glycosyl transferase, phosphoryl transferase, phosphatase, or sialyltransferase); by selective isolation of glycoforms during the α-Gal Apurification process; by treatment of the cell or preparation with acarbohydrate modifying enzyme; or treatment of the cell or preparationwith an inhibitor of glycosylation, e.g., kifunensine, and/or byco-expression in the cell (e.g., via co-transfection) of a DNA encodingany of the foregoing enzymes or other carbohydrate modifying enzymes.

In a preferred embodiment, the method includes the step of analyzing(e.g., assaying) one or more parameters of the carbohydrate signature,biological activity or pharmacokinetic parameter of the α-Gal Apreparation after modification.

The invention also features a method of treating a subject, e.g., ahuman. The method includes: providing or obtaining a panel of two ormore α-Gal A preparations having different glycan characteristics; andselecting α-Gal A preparation having a carbohydrate signature thatmatches one or more (preferably at least two, more preferably at leastthree or more, e.g., at least four, five, six or seven) of theaforementioned parameters (1)-(7) and/or (a)-(c) for treating thesubject.

The method can also include administering one or more doses of atherapeutically effective amount of the selected α-Gal A preparation tothe subject. The subject can be evaluated before, during, and/or afterthe administration. For example, the tissue distribution or serumclearance of the α-Gal A preparation can be evaluated in the subject,e.g., evaluated repeatedly over time. The dose of the preparation canthen be adjusted according to the result of the evaluation. The subjectcan also be evaluated or monitored for status, e.g., clinical status, inresponse to the administration of the α-Gal A preparation.

Different carbohydrate signatures, e.g., each parameter (1) to (7) ordifferent combinations of parameters (1)-(7) can be correlated withhaving desirable pharmacokinetic or other biological properties fordifferent populations, e.g., populations that differ by stage or type ofdisease (e.g., cardiac vs. renal type Fabry disease), age, gender,ethnic background, or genotype.

By “α-Gal A deficiency,” it is meant any deficiency in the amount oractivity of this enzyme in a patient, resulting in abnormalaccumulations of neutral glycolipids (e.g., globotriaosylceramide)primarily in capillary endothelial cells, renal glomerular epithelialcells (podocytes) and glomerular mesangial cells, and/or cardiacmyocytes. The deposits of this material can result in severe neuropathicpain (e.g., acroparasthesia and lacerative pain), serious renal andcardiovascular disease, and/or stroke. The glycolipid accumulation mayinduce severe symptoms as typically observed in males who are sufferingfrom Fabry disease. Alternatively, the accumulation may inducerelatively mild symptoms, as can sometimes be seen in some heterozygousfemale carriers of the defective gene. Affected individuals have agreatly shortened life expectancy; death usually results from renal,cardiac, and/or cerebrovascular complications at approximately thefourth and fifth decade in life.

A “carbohydrate signature” of an α-Gal A preparation is one or moreidentifying characteristic of the glycan structure of a givenpreparation or sample of α-Gal A. The carbohydrate signature can bequalitative or quantitative. For example, a carbohydrate signature of anα-Gal A preparation can include one or more of the following identifyingcharacteristics: (a) the relative level, percentage range or specificvalue of complex vs. high mannose or hybrid glycans; (b) the relativelevel, percentage range or specific value of neutral and sialylated,e.g., mono-sialylated, di-sialylated, tri-sialylated andtetra-sialylated glycan structure; (c) the relative level, percentagerange or specific value of phosphorylated or non-phosphorylated glycans;(d) the relative level, percentage range or specific value of sialylatedglycans; (e) the relative or specific charge profile of the glycans ofthe preparation; (f) the relative or specific ratio of one type ofcharged monosaccharide to another, e.g., the ratio of sialic acid tomannose-6-phosphate; or the ratio of sialylated glycans tophosphorylated glycans.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a representation of the sequence of α-Gal A cDNA, includingthe sequence that encodes the signal peptide (SEQ ID NO:1).

FIG. 2 is a representation of the human α-Gal A amino acid sequence (SEQID NO:2).

FIG. 3A is a schematic map of pGA213C. FIG. 3B is a diagrammaticrepresentation of the targeting construct, pGA213C, and homologousrecombination with the endogenous α-Gal A locus. pGA213C is depicted astargeting sequences aligned above corresponding sequences on theX-chromosomal α-Gal A locus. Positions relative to the methionineinitiation codon, ATG, are indicated by the numbers above the linearmaps. The activation unit containing murine dhfr, bacterial neo, and CMVpromoter/aldolase intron sequences is shown above the position (−221)into which they were inserted by DNA cloning. α-Gal A coding sequencesare indicated by the darkened boxes. α-Gal A non-coding genomicsequences are indicated by the lightly filled boxes. Large arrowheadsindicate the direction of transcription for dhfr and neo expression.

FIG. 4. is a chromatograph showing glycans released from α-Gal A made inhuman cells (Replagal™) vs α-Gal A made in CHO cells (Fabrazyme™). Bothpreparations were analyzed using HPAE-PAD on a Dionex BioLC CarbohydrateSystem. The glycan profiles indicate that there are significantdifferences in the glycan chains of Replagal™ (top) and Fabrazyme™(bottom). Fabrazyme™ is enriched in phosphorylated structures (peakseluting at 65-69 minutes) and more highly sialylated structures(tetra-sialylated structures eluting at 56-60 minutes and tri-sialylatedstructures eluting at 51-55 minutes) as compared to Replagal™ Replagal™is enriched in neutral (peaks at 33-36 minutes), mono-sialylatedstructures (peaks at 39-44 minutes) and di-sialylated structures (peaksat 45-49 minutes).

FIG. 5. is a graph showing internalization of α-Gal A made in humancells (Replagal™) and α-Gal A made in CHO cells (Fabrazyme™) into cells.Normal human fibroblasts were incubated in multi-well culture plates for6 hours in the absence (control, not shown) or presence of Replagal™ orFabrazyme™. This internalization is mannose-6-phosphate inhibitable,indicating that internalization is predominantly via mannose-6-phosphatereceptors. The results indicate that Replagal™ and Fabrazyme™ are notinternalized comparably by the fibroblasts. Fabrazyme™ is cleared morerapidly than Replagal™ by mannose-6-phosphate receptor-mediatedinternalization.

FIG. 6 shows the molecular masses of α-Gal A made in human cells(Replagal™) (top) and α-Gal A made in CHO cells (Fabrazyme™) (bottom) asdetermined by MALDI-TOF mass spectroscopy. The maximum of the majorbroad peak is at 50,755 and 50,705 Da, respectively, consistent with theexpected molecular weight of the glycosylated monomer. A leadingshoulder at approximately 48,071 Da and 47,667 Da is present,representing the lower molecular weight glycoforms for Replagal™ andFabrazyme™, respectively. The leading shoulder, corresponding to thelower molecular weight glycoforms, is much more distinct in the spectrumof Fabrazyme™.

FIG. 7 is a charge profile of the glycans released from α-Gal A made inhuman cells (Replagal™) (bottom) and α-Gal A made in CHO cells(Fabrazyme™) (top). Glycans were derivatized with a fluorescent probeand compared by ion exchange chromatography on a GlycoSep™ C column. Theresults show that Replagal™ has a higher proportion of neutral andmono-charged glycans, and Fabrazyme™ has a higher proportion oftri-charged glycans.

FIG. 8 is a chromatogram of Fabrazyme™ (top) and Replagal™ (bottom) asanalyzed by reversed phase HPLC using a C4 reversed phase column(Vydac). Chromatograms obtained at 214 nm are shown. The leadingshoulder, corresponding to the lower molecular weight glycoforms, ismuch more pronounced in Fabrazyme™:

FIG. 9 is a graph showing serum concentration (U/ml) of α-Gal A(Replagal™) made in human cells from Cynomolgus Monkey dosed IV at 1mg/kg.

FIG. 10 is a graph showing dose proportionality of C_(max) in animalmodels of α-Gal A (Replagal™) made in human cells.

FIG. 11 is a graph showing Replagal™ plasma concentration (U/ml)following infusion at 0.2 mg/kg in a human subject.

FIG. 12 is a graph showing dose proportionality of C_(max) in humans vs.monkey of α-Gal A (Replagal™) made in human cells.

FIG. 13 is a graph showing dose proportionality of area under the curve(AUC) in animal and humans for α-Gal A (Replagal™) made in human cells.

FIG. 14 is a graph showing liver distribution vs. dose in humans of anα-Gal A preparation (Replagal™) made in human cells.

FIG. 15 is a graph showing Replagal™ plasma concentration (U/ml)following infusion at 0.2 mg/kg in a male and female human subject.

FIG. 16 is a schematic drawing of a kit containing an α-Gal Apreparation described herein packaged in a vial and instructions foradministering the preparation.

DETAILED DESCRIPTION OF THE INVENTION Introduction

It has been discovered that human α-Gal A can be made havingmodifications (e.g., in carbohydrate structure, e.g., glycan, phosphateor sialylation modifications) that result in a human α-Gal A preparationhaving pharmacokinetic properties that are desirable for enzymereplacement therapy for α-Gal A deficiency. For example, a preparationof human α-Gal A produced from human cells genetically engineered toproduce human α-Gal A has an exponent “b” for the allometric scalingequation for clearance from the circulation in humans, Y=a(BW)^(b), ofat least 0.85 (preferably up to 0.92), where Y is clearance rate ofα-Gal A (ml/min), “a” is a non-specific constant, and BW is body weight.Such an α-Gal A preparation, as described herein, can be predominantlytaken up by M6P receptors and has a serum clearance less rapid than thatof human α-Gal A produced in non-human cells, e.g., CHO cells.Accordingly, pharmaceutical compositions and kits for treatment of α-GalA deficiency described herein include such a α-Gal A preparations thatare administered in a unit dose substantially smaller than what iscurrently used in the art. For example, in some embodiments, the α-Gal Apreparations described herein are administered in a unit dose of between0.05 mg and 2.0 mg per kilogram of body weight (mg/kg), preferablybetween 0.05 and 5 mg/kg, more preferably between 0.05 and 0.3 mg/kg(e.g., about 0.1, 0.2, 0.25, 0.3, 0.4 or 0.5 mg/kg). The unit dose canbe, e.g., between 0.1×10⁶ U/kg and 10×10⁶ U/kg. In some embodiments, theunit dose of the α-Gal A preparation is between 0.1×10⁶ U/kg and 5×10⁶U/kg, preferably between about 0.1×10⁶ U/kg and 3×10⁶ U/kg. In otherembodiments, the α-Gal A preparations described herein are administeredno more than once every 7 days, e.g., once every 10 days, 14 days or 21days, or once every 4, 5, 6, 7 or 8 weeks. For some patients, even lessfrequent dosing may be possible, e.g., once every 9, 10, 11, 12 weeks ormore.

It is believed that the desirable pharmacokinetics result at least inpart from the glycosylation patterns of the α-Gal A preparation. Theglycosylation patterns required for the desirable pharmacokinetics ofhuman α-Gal A (e.g., at least 50% complex glycans per α-Gal A monomer,on average; a ratio of sialic acid to mannose-6-phosphate (on a mole permole basis) greater than 1.5 to 1, preferably greater than 2 to 1, morepreferably greater than 3 to 1, most preferably greater than 15 to 1 orhigher) can be achieved through a number of methods known in the art.Certain representative embodiments are summarized and described ingreater detail below.

The α-Gal A preparations described herein can be produced in any cell(an α-Gal A production cell) for the treatment of Fabry disease. In someembodiments, the compositions and methods described herein use humanα-Gal A produced using standard genetic engineering techniques (based onintroduction of the cloned α-Gal A gene or cDNA into a host cell), orgene activation, described in more detail below. The human α-Gal A canbe produced in human cells, which provide the carbohydrate modificationsthat are important for the enzyme's pharmacokinetic activity.

However, human α-Gal A can also be produced in non-human cells, e.g.,CHO cells. If the α-Gal A is produced in non-human cells, one or moreof: the α-Gal A expression construct, the non-human cells, or the α-GalA isolated from the non-human cells can be modified, e.g., as describedherein below, to provide α-Gal A preparations having a glycosylationprofile that results in desirable pharmacokinetic properties.

Cells Suitable for Production of Human α-Gal A

Purified human α-Gal A can be obtained from cultured cells, preferablygenetically modified cells, e.g., genetically modified human cells orother mammalian cells, e.g., CHO cells. Insect cells can also be used.

When cells are to be genetically modified for the purposes of treatmentof Fabry disease, the cells may be modified by conventional geneticengineering methods or by gene activation.

According to conventional methods, a DNA molecule that contains an α-GalA cDNA or genomic DNA sequence may be contained within an expressionconstruct and transfected into primary, secondary, or immortalized cellsby standard methods including, but not limited to, liposome-,polybrene-, or DEAE dextran-mediated transfection, electroporation,calcium phosphate precipitation, microinjection, or velocity drivenmicroprojectiles (see, e.g., U.S. Pat. No. 6,048,729, incorporatedherein by reference).

Alternatively, one can use a system that delivers the geneticinformation by viral vector. Viruses known to be useful for genetransfer include adenoviruses, adeno associated virus, herpes virus,mumps virus, pollovirus, retroviruses, Sindbis virus, and vaccinia virussuch as canary pox virus.

Alternatively, the cells may be modified using a gene activation (“GA”)approach, for example, as described in U.S. Pat. No. 5,641,670; U.S.Pat. No. 5,733,761; U.S. Pat. No. 5,968,502; U.S. Pat. No. 6,200,778;U.S. Pat. No. 6,214,622; U.S. Pat. No. 6,063,630; U.S. Pat. No.6,187,305; U.S. Pat. No. 6,270,989; and U.S. Pat. No. 6,242,218, eachincorporated herein by reference. α-Gal A made by gene activation isreferred to herein as GA-GAL (Selden et al., U.S. Pat. Nos. 6,083,725and 6,458,574B1).

Accordingly, the term “genetically modified,” as used herein inreference to cells, is meant to encompass cells that express aparticular gene product following-introduction of a DNA moleculeencoding the gene product and/or including regulatory elements thatcontrol expression of a coding sequence for the gene product. The DNAmolecule may be introduced by gene targeting or homologousrecombination, i.e., introduction of the DNA molecule at a particulargenomic site. Homologous recombination may be used to replace thedefective gene itself (the defective α-Gal A gene or a portion of itcould be replaced in a Fabry disease patient's own cells with the wholegene or a portion thereof).

As used herein, the term “primary cell” includes cells present in asuspension of cells isolated from a vertebrate tissue source (prior totheir being plated, i.e., attached to a tissue culture substrate such asa dish or flask), cells present in an explant derived from tissue, bothof the previous types of cells plated for the first time, and cellsuspensions derived from these plated cells.

“Secondary cells” refers to cells at all subsequent steps in culturing.That is, the first time a plated primary cell is removed from theculture substrate and replated (passaged), it is referred to as asecondary cell, as are all cells in subsequent passages.

A “cell strain” consists of secondary cells which have been passaged oneor more times; exhibit a finite number of mean population doublings inculture; exhibit the properties of contact-inhibited, anchoragedependent growth (except for cells propagated in suspension culture);and are not immortalized.

By “immortalized cell” or “continuous cell-line” is meant a cell from anestablished cell line that exhibits an apparently unlimited lifespan inculture.

Examples of primary or secondary cells include fibroblasts, epithelialcells including mammary and intestinal epithelial cells, endothelialcells, formed elements of the blood including lymphocytes and bonemarrow cells, glial cells, hepatocytes, keratinocytes, muscle cells,neural cells, or the precursors of these cell types. Examples ofimmortalized human cell, lines useful in the present methods include,but are not limited to, Bowes Melanoma cells (ATCC Accession No. CRL9607), Daudi cells (ATCC Accession No. CCL 213), HeLa cells andderivatives of HeLa cells (ATCC Accession Nos. CCL 2, CCL 2.1, and CCL2.2), HL-60 cells (ATCC Accession No. CCL 240), HT-1080 cells (ATCCAccession No. CCL 121), Jurkat cells (ATCC Accession No. TIB 152), KBcarcinoma cells (ATCC Accession No. CCL 17), K-562 leukemia cells (ATCCAccession No. CCL 243), MCF-7 breast cancer cells (ATCC Accession No.BTH 22), MOLT-4 cells (ATCC Accession No. 1582), Namalwa cells (ATCCAccession No. CRL 1432), Raji cells (ATCC Accession No. CCL 86), RPMI8226 cells (ATCC Accession No. CCL 155), U-937 cells (ATCC Accession No.CRL 15 93), WI-3 8VAI 3 sub line 2R4 cells (ATCC Accession No. CLL75.1), CCRF-CEM cells (ATCC Accession No. CCL 119), and 2780AD ovariancarcinoma cells (Van der Blick et al., Cancer Res. 48: 5927-5932, 1988),as well as heterohybridoma cells produced by fusion of human cells andcells of another species.

Following the genetic modification of human cells to produce a cellwhich secretes α-Gal. A, a clonal cell strain consisting essentially ofa plurality of genetically identical cultured primary human cells or,where the cells are immortalized, a clonal cell line consistingessentially of a plurality of genetically identical immortalized humancells, may be generated. In one embodiment, the cells of the clonal cellstrain or clonal cell line are fibroblasts. In a preferred embodimentthe cells are secondary human fibroblasts, e.g., BRS-11 cells. Example 1provides additional guidance on the production of cells geneticallyengineered to produce human α-Gal A.

After genetic modification, the cells are cultured under conditionspermitting production and secretion of α-Gal A. The protein is isolatedfrom the cultured cells by collecting the medium in which the cells aregrown, and/or lysing the cells to release their contents, and thenapplying protein purification techniques.

Increasing Circulatory Half Life, Cellular Uptake and/or Targeting ofα-Gal A to Appropriate Tissues

The data described herein shows that human α-Gal A can be made havingmodifications (e.g., carbohydrate, phosphate or sialylationmodifications) that result in pharmacokinetic properties of the enzymethat are desirable for use in enzyme replacement therapy for α-Gal Adeficiency. One method of making such human α-Gal A preparations is toproduce human α-Gal A from human cells.

There are differences in the glycosylation characteristics of human andnon-human cells (e.g., CHO cells) such that the production of α-Gal A(or indeed, of any glycoprotein) from human cells necessarily results ina structurally different protein than that produced in CHO cells.Although not bound by theory, these differences are thought to beimportant for the desirable pharmacokinetics of human α-Gal Apreparations in the compositions and methods described herein. However,α-Gal A preparations described herein can also be produced fromnon-human cells, wherein either the cells, the α-Gal A coding sequenceand/or the purified α-Gal A are modified. For example, non-human cellswhose glycosylation machinery differs from human (e.g., CHO cells) canbe genetically modified to express an enzyme of carbohydrate metabolism,e.g., α-2,6-sialyltransferase, that is present in human but not in CHOcells.

In another example, the cells can be genetically engineered to expressan α-Gal A protein that has one or more modified glycosylation sites,e.g., a human or non-human cell can be genetically engineered to expressan α-Gal A coding sequence in which one or more additional N-linkedglycosylation sites have been added or deleted. The additionalglycosylation sites can be glycosylated by the cellular machinery in thecell, e.g., the CHO cell, in which the modified α-Gal A coding sequenceis expressed, thus providing an α-Gal A preparation that has anincreased circulatory half-life, cellular uptake, and/or improvedtargeting to heart, kidney or other appropriate tissues compared to theunmodified α-Gal A, e.g., when expressed in non-human cells.

α-Gal A can also be modified (e.g., after isolation from a geneticallyengineered non-human cell) to resemble human α-Gal A produced in humancells. For example, a human α-Gal A preparation isolated from anon-human cell can be modified, e.g., phosphorylated or cleaved (e.g.,with neuraminidase or phosphatase) before administration to a subject.

The circulating half-life, cellular uptake and/or tissue targeting canalso be modified, inter alia, by (i) modulating the phosphorylation ofα-Gal A; (ii) modulating the sialic acid content of α-Gal A; and/or(iii) sequential removal of the sialic acid and terminal galactoseresidues, or removal of terminal galactose residues, on theoligosaccharide chains on α-Gal A. Altered sialylation of α-Gal Apreparations can enhance the circulatory half-life, cellular uptakeand/or tissue targeting of exogenous α-Gal A. A change in the ratio ofmoles of mannose-6-phosphate per mole of sialic acid per molecule ofα-Gal A can also result in improved cellular uptake, relative to that ofhepatocytes, in non-hepatocytes such as liver endothelial cells, liversinusoidal cells, capillary/vascular endothelial cells, renal glomerularepithelial cells (podocytes) and glomerular mesangial cells, renalendothelial cells, pulmonary cells, renal cells, neural cells, and/orcardiac myocytes. For example, a preferred ratio of sialic acid tomannose-6-phosphate in the α-Gal A preparation (on a mole per molebasis) is greater than 1.5 to 1, preferably greater than 2 to 1, morepreferably greater than 3 to 1, most preferably greater than 3.5 to 1 orhigher.

Glycan Remodeling

Glycoprotein modification (e.g., when α-Gal A is produced in non-humancells) can increase uptake of the enzyme in specific tissues other thanliver and macrophages, e.g., increase uptake in capillary/vascularendothelial cells, renal glomerular epithelial cells (podocytes) andglomerular mesangial cells, renal endothelial cells, pulmonary cells,renal cells, neural cells, and/or cardiac myocytes. Using glycoproteinmodification methods, human glycosylated α-Gal A preparations can beobtained, wherein between 35% and 85% of the oligosaccharides,preferably at least 50%, are charged.

Protein N-glycosylation functions by modifying appropriate asparagineresidues of proteins with oligosaccharide structures, thus influencingtheir properties and bioactivities (Kukuruzinska & Lennon, Crit. Rev.Oral. Biol. Med. 9: 415-48 (1998)). An α-Gal A preparation describedherein can have a high percentage of the oligosaccharides beingnegatively charged, primarily by the addition of one to four sialic acidresidues on complex glycans, or of one to two phosphate moieties onhigh-mannose glycans, or of a single phosphate and a single sialic acidon hybrid glycans. Smaller amounts of sulfated complex glycans may alsobe present. A high proportion of charged structures serves two mainfunctions. First, capping of penultimate galactose residues by 2,3- or2,6-linked sialic acid prevents premature removal from the circulationby the asialoglycoprotein receptor present on hepatocytes. This receptorrecognizes glycoproteins with terminal galactose residues.

Modifying the glycosylation pattern of α-Gal A produced in non-humancells to, e.g., resemble the pattern produced in human cells, givesimportant target organs such as heart and kidney the opportunity toendocytose greater amounts of enzyme from the plasma following enzymeinfusion. Second, the presence of Man-6-phosphate on high-mannose orhybrid glycans provides an opportunity for receptor-mediated uptake bythe cation-independent Man-6-phosphate receptor (CI-MPR). Thisreceptor-mediated uptake occurs on the surface of many cells, includingvascular endothelial cells, which are a major storage site of Gb₃ inFabry patients. Enzyme molecules with two Man-6-phosphate residues havea much greater affinity for the CI-MPR than those with a singleMan-6-phosphate.

The complexity of N-glycosylation is augmented by the fact thatdifferent asparagine residues within the same polypeptide may bemodified with different oligosaccharide structures, and various proteinsare distinguished from one another by the characteristics of theircarbohydrate moieties.

Several approaches are provided herein for carbohydrate remodeling on aprotein containing N-linked glycan chains. First, one can geneticallyengineer a cell, e.g., a non-human cell, to produce a human α-Gal Ahaving a non-naturally occurring glycosylation site, e.g., one canengineer a human α-Gal A coding sequence to produce an α-Gal A proteinhaving one or more additional glycosylation sites. The additionalglycosylation sites can be glycosylated (e.g., with complex glycans) bythe cellular machinery in the cell, e.g., the CHO cell, in which themodified α-Gal A coding sequence is expressed, thus providing an α-Gal Apreparation that has improved circulatory half-life, cellular uptakeand/or tissue targeting compared to the unmodified α-Gal A, e.g., whenexpressed in non-human cells.

Second, the proportion of charged α-Gal A can be increased by selectiveisolation of glycoforms during the purification process. The presentinvention provides for increasing the proportion of highly charged andhigher molecular weight α-Gal A glycoforms by fractionation of α-Gal Aspecies on chromatography column resins during and/or after thepurification process. The more highly charged glycoform species of α-GalA contain more sialic acid and/or more phosphate, and the highermolecular weight glycoforms would also contain the fully glycosylated,most highly branched and highly charged species. Selection of thecharged species, or removal of the non-glycosylated, poorly glycosylatedor poorly sialylated and/or phosphorylated α-Gal A species would resultin a population of α-Gal A glycoforms with more sialic acid and/or amore desirable sialic acid to phosphate ratio in the preparation,therefore providing an α-Gal A preparation with better half-life,cellular uptake and/or tissue targeting, thereby having bettertherapeutic efficiency.

This fractionation process can occur on, but is not limited to, suitablechromatographic column resins utilized to purify or isolate α-Gal A. Forexample, fractionation can occur on, but is not limited to, cationexchange resins (such as SP-SepharoseG), anion exchange resins(Q-SepharoseG), affinity resins (Heparin Sepharose-b, lectin columns)size exclusion columns (Superdex 200) and hydrophobic interactioncolumns (Butyl Sepharose) and other chromatographic column resins knownin the art.

Since α-Gal A is produced in cells as a heterogeneous mixture ofglycoforms which differ in molecular weight and charge, α-Gal A tends toelute in relatively broad peaks from the chromatography resins. Withinthese elutions, the glycoforms are distributed in a particular mannerdepending on the nature of the resin being utilized. For example, onsize exclusion chromatography, the largest glycoform will tend to eluteearlier on the elution profile than the smaller glycoforms. On ionexchange chromatography, the most negatively charged glycoforms willtend to bind to a positively charged resin (such as Q-Sepharoseg) withhigher affinity than the less negatively charged glycoforms, and willtherefore tend to elute later in the elution profile. In contrast, thesehighly negatively charged glycoforms may bind less tightly to anegatively charged resin, such as SP Sepharose8, than less negativelycharges species, or may not even bind at all.

Fractionation and selection of highly charged and/or higher molecularweight glycoforms of α-Gal A can be performed on any α-Gal Apreparation, such as that derived from genetically modified cells suchas cells, e.g., human or non-human cells, modified by conventionalgenetic engineering methods or by gene activation (GA). It can beperformed on cell lines grown in optimized systems to provide alteredsialylation and phosphorylation as described herein, e.g., to provide apreparation with a ratio of sialic acid to mannose-6-phosphate (on amole per mole basis) is greater than 1.5 to 1, preferably greater than 2to 1, more preferably greater than 3 to 1, most preferably greater than3.5 to 1 or higher.

A third approach for carbohydrate remodeling can involve modifyingcertain glycoforms on the purified α-Gal A by attachment of anadditional terminal sugar residue using a purified glycosyl transferaseand the appropriate nucleotide sugar donor. This treatment affects onlythose glycoforms that have an appropriate free terminal sugar residue toact as an acceptor for the glycosyl transferase being used. For example,a 2,6-sialyltransferase adds sialic acid in an α-2,6-linkage onto aterminal Galβ1,4GlcNAc-R acceptor, using CMP-sialic acid as thenucleotide sugar donor. Commercially available enzymes and their speciesof origin include: fucose α 1,3 transferases III, V and VI (humans);galactose α 1,3 transferase (porcine); galactose β1,4 transferase(bovine); mannose α1,2 transferase (yeast); sialic acid α 2,3transferase (rat); and sialic acid α 2,6 transferase (rat). After thereaction is completed, the glycosyl transferase can be removed from thereaction mixture by a glycosyl transferase specific affinity columnconsisting of the appropriate nucleotide bonded to a gel through a 6carbon spacer by a pyrophosphate (GDP, UDP) or phosphate (CMP) linkageor by other chromatographic methods known in the art. Of the glycosyltransferases listed above, the sialyl transferases is particularlyuseful for modification of enzymes, such as α-Gal A, for enzymereplacement therapy in human patients. Use of either sialyl transferasewith CMP-5-fluoresceinyl-neuraminic acid as the nucleotide sugar donoryields a fluorescently labeled glycoprotein whose uptake and tissuelocalization can be readily monitored.

A fourth approach for carbohydrate remodeling involvesglyco-engineering, e.g., introduction of genes that affect glycosylationmechanisms of the cell, of the α-Gal A production cell to modifypost-translational processing in the Golgi apparatus is a preferredapproach.

A fifth approach for carbohydrate remodeling involves treating α-Gal Awith appropriate glycosidases to reduce the number of differentglycoforms present. For example, sequential treatment of complex glycanchains with neuraminidase, β-galactosidase, and β-hexosaminidase cleavesthe oligosaccharide to the trimannose core.

A sixth approach for glycan remodeling involves the use of inhibitors ofglycosylation, e.g., kifunensine (an inhibitor of mannosidase I),swainsonine, or the like. Such inhibitors can be added to the culturedcells expressing a human α-Gal A. The inhibitors are taken up into thecells and inhibit glycosylation enzymes, such as glycosyl transferasesand glycosidases, providing α-Gal A molecules with altered sugarstructures. Alternatively, a cell genetically engineered to producehuman α-Gal A can be transfected with glycosylation enzymes such asglycosyl transferases and glycosidases.

A seventh approach involves using glycosylation enzymes (e.g., glycosyltransferases or glycosidases) to remodel the carbohydrate structures invitro, e.g., on an α-Gal A that has been isolated from a geneticallyengineered cell, as described herein.

Other approaches for glycan remodeling are known in the art.

Altering Half Life and/or Cellular Uptake of α-Gal A by AlteringSialylation

Sialylation affects the circulatory half-life and biodistribution ofproteins. Proteins with minimal or no sialic acid are readilyinternalized by the asialoglycoprotein receptor (Ashwell receptor) onhepatocytes by exposed galactose residues on the protein. Thecirculating half-life of galactose-terminated α-Gal A can be altered bysequentially (1) removing sialic acid by contacting α-Gal A withneuraminidase (sialidase), thereby leaving the terminal galactosemoieties exposed, and (2) removing the terminal galactoside residues bycontacting the desialylated α-Gal A with β-galactosidase. The resultingα-Gal A preparation has a reduced number of terminal sialic acid and/orterminal galactoside residues on the oligosaccharide chains compared toα-Gal A preparations not sequentially contacted with neuraminidase andβ-galactosidase. Alternatively, the circulating half-life ofgalactose-terminated α-Gal A can be enhanced by only removing theterminal galactoside residues by contacting the desialylated α-Gal. Awith β-galactosidase. The resulting α-Gal A preparation has a reducednumber of terminal galactoside residues on the oligosaccharide chainscompared to α-Gal A preparations not contacted with β-galactosidase. Ina preferred embodiment, following sequential contact with neuraminidaseand β-galactosidase, the resulting α-Gal A preparations are subsequentlycontacted with β-hexosaminidase, thereby cleaving the oligosaccharide tothe trimannose core.

The sialic acid content of α-Gal A preparations can be increased by (i)isolation of the highly charged and/or higher molecular weight α-Gal Aglycoforms during or after the purification process; (ii) adding sialicacid residues using cells genetically modified (either by conventionalgenetic engineering methods or gene activation) to express a sialyltransferase gene or cDNA; or (iii) fermentation or growth of cellsexpressing the enzyme in a low ammonium environment.

Altering Half Life and/or Cellular Uptake by Altering Phosphorylation

Altering the phosphorylation of an α-Gal A preparation described hereincan alter the circulatory half life and cellular uptake of thepreparation into desired tissues. In preferred embodiments, an α-Gal Apreparation has less than 45% phosphorylated glycans. For example, thepreparation has less than about 35%, 30%, 25%, or 20% phosphorylatedglycans. A desirable ratio of sialic acid mannose-6-phosphate in theα-Gal A preparation (on a mole per mole basis) is a ratio greater than1.5 to 1, preferably greater than 2 to 1, more preferably greater than 3to 1, most preferably greater than 3.5 to 1 or higher.

The phosphorylation of α-Gal A preparations can be modified, e.g.,increased or decreased, by (i) adding or removing phosphate residuesusing cells genetically modified (either by conventional geneticengineering methods or gene activation) to express a phosphoryltransferase or phosphatase gene or cDNA; (ii) adding phosphatases,kinases, or their inhibitors to the cultured cells; or (iii) addingphosphatases; kinases, or their inhibitors to a purified α-Gal Apreparation produced from a genetically engineered cell as describedherein.

The concerted actions of two membrane-bound Golgi enzymes are needed togenerate a Man-6-phosphate recognition marker on a lysosomal proenzyme.The first, UDP-N-acetylglucosamine: glycoproteinN-acetylglucosamine-1-phosphotransferase (GlcNAc phosphotransferase),requires a protein recognition determinant on lysosomal enzymes thatconsists of two lysine residues 34 Å apart and in the correct spatialrelationship to a high mannose chain. The second,N-acetylglucosamine-1-phosphodiester a-N-acetylglucosaminidase(phosphodiester α-GlcNAcase), hydrolyzes the α-GlcNAc-phosphate bondexposing the Man-6-phosphate recognition site. These enzymes can beinduced or inhibited by methods known in the art to provide an α-Gal Apreparation with desirable phosphorylation characteristics (e.g., with adesirable ration of sialylated to phosphorylated glycans).

In one embodiment, an α-Gal A preparation with altered phosphorylationis obtained by first introducing into an α-Gal A production cell apolynucleotide which encodes for phosphoryl transferase, or byintroducing a regulatory sequence by homologous recombination thatregulates expression of an endogenous phosphoryl transferase gene. Theα-Gal A production cell is then cultured under culture conditions whichresults in expression of α-Gal A and phosphoryl transferase. The α-Gal Apreparation with increased phosphorylation compared to the α-Gal Aproduced in a cell without the polynucleotide is then isolated.

In still another embodiment, a glycosylated α-Gal A preparation withaltered phosphorylation is obtained by adding a phosphatase inhibitor,e.g., bromotetramisole, or a kinase inhibitor, to cultured cells.

Using the methods described herein, α-Gal A preparations are obtainedwherein at doses below serum or plasma clearance saturation levels,serum clearance of the α-Gal A preparation from the circulation ispreferably less than 4 mL/min/kg on the linear portion of the AUC vs.dose curve, more preferably less than about 3.5, 3, or 2.5 mL/min/kg, onthe linear portion of the AUC vs. dose curve. The α-Gal A preparationhas an exponent “b” for the allometric scaling equation for clearancefrom the circulation in mammals, Y=(BW)^(b), of at least 0.85, where Yis clearance of α-Gal A from the circulation (ml/min), “a” is anon-specific constant and BW is body weight. The exponent “b” ispreferably at least 0.88, more preferably at least 0.90, and mostpreferably at least 0.92, 0.94 or higher.

In preferred embodiments, an α-Gal A preparation described herein isenriched in neutral, mono-sialylated and di-sialylated glycan structures(combined) relative to more highly sialylated structures such astri-sialylated and tetra-sialylated structures. For example, a preferredα-Gal A preparation has one or more of: (a) at least about 22% neutralglycans, e.g., at least about 25% or 30% neutral glycans; (b) at leastabout 15%, 20%, or 25% mono-sialylated glycans; (c) at least about 35%,preferably at least about 40%, 45%, or 50% neutral and mono-sialylatedglycans combined; (d) at least about 75%, 76%, 78% or more neutral,mono- and di-sialylated glycans combined; and (e) less than about 35%,preferably less than about 25%, 20%, 18% or about 15% tri- andtetra-sialylated glycan structures combined.

In preferred embodiments, an α-Gal A preparation described herein has,on average, more than one complex glycan per monomer, preferably atleast 50% complex glycans per monomer, e.g., 2 complex glycans or moreper monomer.

In preferred embodiments, an α-Gal A preparation described herein has atleast 5%, preferably at least 7%, 10% or 15% neutral glycans.

In preferred embodiments, an α-Gal A preparation described herein hasless than 45% phosphorylated glycans. For example, the preparation hasless than about 35%, 30%, 25%, or 20% phosphorylated glycans.

In preferred embodiments, an the α-Gal A preparation described hereinhas a total proportion of sialylated glycans greater than about 45%,e.g., greater than 50% or 55%.

In a preferred embodiment, the ratio of sialic acid tomannose-6-phosphate in the α-Gal A preparation (on a mole per molebasis) is greater than 1.5 to 1, preferably greater than 2 to 1, morepreferably greater than 3 to 1, most preferably greater than 3.5 to 1 orhigher.

In one embodiment, the percent ratio of sialylated glycans tophosphorylated glycans is greater than 1, preferably greater than 1.5,more preferably greater than 2, e.g., greater than about 2.5 or 3.

PEGylation

In other embodiments, the circulatory half-life of a human α-Gal Apreparation is enhanced by complexing α-Gal A with polyethylene glycol(PEG). In a preferred embodiment, the α-Gal A preparation is complexedusing tresyl monomethoxy PEG (TMPEG) to form a PEGylated-α-Gal A. ThePEGylated-α-Gal A is then purified, to provide an isolated,PEGylated-α-Gal A preparation. PEGylation of α-Gal A increases thecirculating half-life, cellular uptake and/or tissue distribution of theprotein.

Purification of α-Gal A from the Conditioned Medium of StablyTransfected Cells

α-Gal A may be purified to near-homogeneity from the cultured cellstrains and/or conditioned medium of the cultured cell strains that havebeen stably transfected to produce the enzyme. α-Gal A can be isolatedfrom α-Gal A containing media using chromatographic steps. For example,1 or more, e.g., 2, 3, 4, 5 or more chromatographic steps can be used.The different steps of chromatography utilize various separationprinciples which take advantage of different physical properties of theenzyme to separate α-Gal A from contaminating material. For example, thesteps can include: hydrophobic interaction chromatography on butylSepharose, ionic interaction on hydroxyapatite, anion exchangechromatography on Q Sepharose and size exclusion chromatography onSuperdex 200. Size exclusion chromatography can serve as an effectivemeans to exchange the purified protein into a formulation-compatiblebuffer.

One purification process includes the use of butyl Sepharose®chromatography as a first step in purification. Other hydrophobicinteraction resins, such as Source Iso (Pharmacia), Macro-Prep® MethylSupport (Bio-Rad), TSK Butyl (Tosohaas) or Phenyl Sepharose® (Pharmacia)can also be used. The column can be equilibrated in a relatively highconcentration of a salt, e.g., 1 M ammonium sulfate or 2 M sodiumchloride, e.g., in a buffer of pH 5.6. The sample to be purified can beprepared by adjusting the pH and salt concentration to those of theequilibration buffer. The sample is applied to the column and the columnis washed with equilibration buffer to remove unbound material. Theα-Gal A is eluted from the column with a lower ionic strength buffer,water, or organic solvent in water, e.g., 20% ethanol or 50% propyleneglycol. Alternatively, the α-Gal A can be made to flow through thecolumn by using a lower concentration of salt in the equilibrationbuffer and in the sample or by using a different pH. Other proteins maybind to the column, resulting in purification of the α-Gal A-containingsample which did not bind the column.

An alternative step of purification can use a cation exchange resin,e.g., SP Sepharose® 6 Fast Flow (Pharmacia), Source 30S (Pharmacia), CMSepharose® Fast Flow (Pharmacia), Macro-Prep® CM Support (Bio-Rad) orMacro-Prep® High S Support (Bio-Rad), to purify α-Gal A. The “firstchromatography step” is the first application of a sample to achromatography column (all steps associated with the preparation of thesample are excluded). The α-Gal A can bind to the column at pH 4.4. Abuffer, such as 10 mM sodium acetate, pH 4.4, 10 mM sodium citrate, pH4.4, or other buffer with adequate buffering capacity at approximatelypH 4.4, can be used to equilibrate the column. The sample to be purifiedis adjusted to the pH and ionic strength of the equilibration buffer.The sample is applied to the column and the column is washed after theload to remove unbound material. A salt, such as sodium chloride orpotassium chloride, can be used to elute the α-Gal A from the column.Alternatively, the α-Gal. A can be eluted from the column with a bufferof higher pH or a combination of higher salt concentration and higherpH. The α-Gal A can also be made to flow through the column duringloading by increasing the salt concentration in the equilibration bufferand in the sample load, by running the column at a higher pH, or by acombination of both increased salt and higher pH.

Another step of purification can use a Q Sepharose® 6 Fast Flow for thepurification of α-Gal A. Q Sepharose® 6 Fast Flow is a relatively stronganion exchange resin. A weaker anion exchange resin such as DEAESepharose® Fast Flow (Pharmacia) or Macro-Prep® DEAR (Bio-Rad) can alsobe used to purify α-Gal A. The column is equilibrated in a buffer, e.g.,10 mM sodium phosphate, pH 6. The pH of the sample is adjusted to pH 6,and low ionic strength is obtained by dilution or diafiltration of thesample. The sample is applied to the column under conditions that bindα-Gal A. The column is washed with equilibration buffer to removeunbound material. The α-Gal A is eluted with application of salt, e.g.,sodium chloride or potassium chloride, or application of a lower pHbuffer, or a combination of increased salt and lower pH. The α-Gal A canalso be made to flow through the column during loading by increasing thesalt concentration in the load or by running the column at a lower pH,or by a combination of both increased salt and lower pH.

Another step of purification can use a Superdex® 200 (Pharmacia) sizeexclusion chromatography for purification of α-Gal A. Other sizeexclusion chromatography resins such as Sephacryl® S-200 HR or Bio-Gel®A-1.5 m can also be used to purify α-Gal A. The preferred buffer forsize exclusion chromatography is 25 mm sodium phosphate, pH 6.0,containing 0.15 M sodium chloride. Other formulation-compatible bufferscan also be used, e.g., 10 mM sodium or potassium citrate. The pH of thebuffer can be between pH 5 and pH 7 and should at contain a salt, e.g.,sodium chloride or a mixture of sodium chloride and potassium chloride.

Another step of purification can use a chromatofocusing resin such asPolybuffer Exchanger PBE 94 (Pharmacia) to purify α-Gal A. The column isequilibrated at relatively high pH (e.g., pH 7 or above), the pH of thesample to be purified is adjusted to the same pH, and the sample isapplied to the column. Proteins are eluted with a decreasing pH gradientto a pH such as pH 4, using a buffer system, e.g., Polybuffer 74(Pharmacia), which had been adjusted to pH 4.

Alternatively, immunoaffinity chromatography can be used to purify α-GalA. An appropriate polyclonal or monoclonal antibody to α-Gal A(generated by immunization with α-Gal A or with a peptide derived fromthe α-Gal A sequence using standard techniques) can be immobilized on anactivated coupling resin, e.g., NHS-activated Sepharose® 4 Fast Flow(Pharmacia) or CNBr-activated Sepharose® 4 Fast Flow (Pharmacia). Thesample to be purified can be applied to the immobilized antibody columnat about pH 6 or pH 7. The column is washed to remove unbound material.α-Gal A is eluted from the column with typical reagents utilized foraffinity column elution such as low pH, e.g., pH 3, denaturant, e.g.,guanidine HCl or thiocyanate, or organic solvent, e.g., 50% propyleneglycol in a pH 6 buffer. The purification procedure can also use a metalchelate affinity resin, e.g., Chelating Sepharose® Fast Flow(Pharmacia), to purify α-Gal A. The column is pre-charged with metalions, e.g., Cu⁺², Zn⁺², Ca⁺², Mg⁺² of Cd⁺². The sample to be purified isapplied to the column at an appropriate pH, e.g., pH 6 to 7.5, and thecolumn is washed to remove unbound proteins. The bound proteins areeluted by competitive elution with imidazole or histidine or by loweringthe pH using sodium citrate or sodium acetate to a pH less than 6, or byintroducing chelating agents, such as EDTA or EGTA.

Dosages for Administration of α-Gal A Preparation

The α-Gal A preparations described herein exhibit a desirablecirculatory half-life and tissue distribution, e.g., to capillaryendothelial cells, renal glomerular epithelial cells (podocytes) andglomerular mesangial cells, and/or cardiac myocytes. Such preparationscan be administered in relatively low dosages. For example, the unitdose of administration can be between 0.05-2.0 mg per kilogram bodyweight (mg/kg). For example, the unit dose can be between 0.05 and 1.0mg, between 0.5 and 0.5 mg/kg, or between 0.5 and 0.3 mg/kg. Unit dosesbetween 0.05 and 0.29 mg/kg are preferred, e.g., a unit dose of about0.05, 0.1, 0.15, 0.2, 0.25, mg/kg. Assuming a specific activity of theα-Gal A preparation of between 2 and 4.5×10⁶ U/mg, these valuescorrespond to about 0.1×10⁶ to 1.3×10⁶ U/kg. A preferred unit dosesaturates liver uptake of the α-Gal A.

Regularly repeated doses of the protein are necessary over a period oftime, e.g., for a period of several months or 1, 2, 3 years or longer,even for the life of the patient. However, the desirable circulatoryhalf-life and tissue distribution of the α-Gal A preparations describedherein allow for the administration of the unit dose to a patient atrelatively long intervals. For example, a unit dose can be administeredno more than once every 7 days, 10 days, 14 days, 21 days, 4 weeks, 6weeks, 8 weeks, 10 weeks or 12 weeks. A preferred frequency of dosing isbiweekly, monthly or bimonthly.

During the time of therapy, a patient can be monitored clinically toevaluate the status of his or her disease. Clinical improvement measuredby, for example, improvement in renal or cardiac function or patient'soverall well-being (e.g., pain), and laboratory improvement measured by,for example, reductions in urine, plasma, or tissue Gb₃ levels, may beused to assess the patient's health status. In the event that clinicalimprovement is observed after a treatment and monitoring period, thefrequency of α-Gal A administration may be reduced. For example, apatient receiving weekly injections of α-Gal A preparation may change tobiweekly administration; a patient receiving biweekly injections of anα-Gal A preparation may switch to monthly administration; a patientreceiving monthly injections of an α-Gal A preparation may switch tobi-monthly injections. Following such a change in dosing frequency, thepatient should be monitored for another period of time, e.g., severalyears, e.g., a three year period, in order to assess Fabrydisease-related clinical and laboratory measures. In a preferredembodiment, the administered dose does not change if a change in dosingfrequency is made. This ensures that certain pharmacokinetic parameters(e.g. maximal plasma concentration [C_(max)], time to maximal plasmaconcentration [t_(max)], plasma, half-life [t_(1/2)], and exposure asmeasured by area under the curve [AUC]) remain relatively constantfollowing each administered dose. Maintenance of these pharmacokineticparameters will result in relatively constant levels ofreceptor-mediated uptake of α-Gal A into tissues as dose frequencieschange.

In some embodiments, a patient is clinically evaluated between doses anda determination can be made upon evaluation as to the timing of the nextdose.

Subcutaneous injections can be used to maintain longer term exposure tothe drug. Dosages of the α-Gal A preparations that are administered byintramuscular injections may be the same or different than thoseinjected subcutaneously. In a preferred embodiment, intramusculardosages are smaller and administered less frequently. The α-Gal Apreparation is preferably administered intravenously, e.g., in aintravenous bolus injection, in a slow push intravenous injection, or bycontinuous intravenous injection. Continuous IV infusion (e.g., over 2-6hours) allows the maintenance of specific levels in the blood.

A patient with atypical variant of Fabry disease, e.g., exhibitingpredominantly cardiovascular abnormalities or renal involvement, can betreated with these same dosage regiments as described herein. The doseis adjusted as needed. For example, a patient with the cardiac variantphenotype who is treated with α-Gal A enzyme replacement therapy willhave a change in the composition of their heart and improved cardiacfunction following therapy. This change can be measured with standardechocardiography which is able to detect increased left ventricular wallthickness in patients with Fabry disease (Goldman et al., JAm CollCardiol 7: 1157-1161 (1986)). Serial echocardiographic measurements ofleft ventricular wall thickness can be conducted during therapy, and adecrease in ventricular wall size is indicative of a therapeuticresponse. Patients undergoing α-Gal A enzyme replacement therapy canalso be followed with cardiac magnetic resonance imaging (MRI). MRI hasthe capability to assess the relative composition of a given tissue. Forexample, cardiac MRI in patients with Fabry disease reveals depositedlipid within the myocardium compared with control patients (Matsui etal., Ani Heart J 117: 472-474. (1989)). Serial cardiac MRI evaluationsin a patient undergoing enzyme replacement therapy can reveal a changein the lipid deposition within a patient's heart. Patients with therenal variant phenotype can also benefit from α-Gal A enzyme replacementtherapy.

The effect of therapy can be measured by standard tests of renalfunction, such as 24-hour urine protein level, creatinine clearance, andglomerular filtration rate.

Pharmaceutical Compositions

The α-Gal A preparations described herein are substantially free ofnon-α-Gal A proteins, such as albumin, non-α-Gal A proteins produced bythe host cell; or proteins isolated from animal tissue or fluid. Thepreparation preferably comprises part of an aqueous or physiologicallycompatible fluid suspension or solution. The carrier or vehicle isphysiologically compatible so that, in addition to delivery of thedesired preparation to the patient, it does not otherwise adverselyaffect the patient's electrolyte and/or volume balance. Useful solutionsfor parenteral administration may be prepared by any of the methods wellknown in the pharmaceutical art (See; e.g., REMINGTON'S PHARMACEUTICALSCIENCES Gennaro, A., ed., Mack Pub., 1990).

Non-parenteral formulations, such as suppositories and oralformulations, can also be used. Preferably the formulation contains anexcipient. Pharmaceutically acceptable excipients for α-Gal A which maybe included in the formulation are buffers such as citrate buffer,phosphate buffer, acetate buffer, and bicarbonate buffer, amino acids,urea, alcohols, ascorbic acid, phospholipids; proteins, such as serumalbumin, collagen, and gelatin; salts such as EDTA or EGTA, and sodiumchloride; liposomes; polyvinylpyrollidone; sugars, such as dextran,mannitol, sorbitol, and glycerol; propylene glycol and polyethyleneglycol (PEG); glycerol; glycine or other amino acids; and lipids.Preferred excipients include mannitol, sorbitol, glycerol, amino acids,lipids, EDTA, EGTA, sodium chloride, polyethylene glycol,polyvinylpyrollidone, dextran, or combinations of any of theseexcipients.

In another embodiment, the formulation further comprises a non-ionicdetergent. Preferred non-ionic detergents include Polysorbate 20,Polysorbate 80, Triton X-100™, Triton X-114™, Nonidet P-40™, Octylα-glucoside, Octyl β-glucoside, Brij 35, Pluronic™, Poloxamer 188(a.k.a. Poloxalkol) and Tween 20™. In a preferred embodiment, thenon-ionic detergent comprises Polysorbate 20 or Polysorbate 80.

A preferred formulation further comprises phosphate-buffered saline,e.g., at pH 6. Buffer systems for use with α-Gal A preparations includecitrate; acetate; bicarbonate; and phosphate buffers (all available fromSigma). Phosphate buffer is a preferred embodiment. A preferred pH rangefor α-Gal A preparations is pH 4.5-7.4.

For lyophilization of α-Gal A preparations, the protein concentrationcan be 0.1-10 mg/mL. Bulking agents, such as glycine, mannitol, albumin,and dextran, can be added to the lyophilization mixture. In addition,possible cryoprotectants, such as disaccharides, amino acids, and PEG,can be added to the lyophilization mixture. Any of the buffers,excipients, and detergents listed above, can also be added.

Formulations for administration may include glycerol and othercompositions of high viscosity to help maintain the agent at the desiredlocus. Biocompatible polymers, preferably bioresorbable, biocompatiblepolymers (including, e.g., hyaluronic acid, collagen, polybutyrate,lactide, and glycolide polymers and lactide/glycolide copolymers) may beuseful excipients to control the release of the agent in vivo.Formulations for parenteral administration may include glycocholate forbuccal administration, methoxysalicylate for rectal administration, orcutnic acid for vaginal administration. Suppositories for rectaladministration may be prepared by mixing an α-Gal A preparation of theinvention with a non-irritating excipient such as cocoa butter or othercompositions that are solid at room temperature and liquid at bodytemperatures.

Formulations for inhalation administration may contain lactose or otherexcipients, or may be aqueous solutions which may containpolyoxyethylene-9-lauryl ether, glycocholate or deoxycocholate. Apreferred inhalation aerosol is characterized by having particles ofsmall mass density and large size. Particles with mass densities lessthan 0.4 gram per cubic centimeter and mean diameters exceeding 5 μmefficiently deliver inhaled therapeutics into the systemic circulation.Such particles are inspired deep into the lungs and escape the lungs'natural clearance mechanisms until the inhaled particles deliver theirtherapeutic payload. (Edwards et al., Science 276: 1868-1872 (1997)).α-Gal A preparations of the present invention can be administered inaerosolized form, for example by using methods of preparation andformulations as described in U.S. Pat. Nos. 5,654,007; 5,780,014, and5,814,607, each incorporated herein by reference.

Formulation for intranasal administration may include oily solutions foradministration in the form of nasal drops, or as a gel to be appliedintranasally.

Formulations for topical administration to the skin surface may beprepared by dispersing the α-Gal A preparation with a dermatologicalacceptable carrier such as a lotion, cream, ointment, or soap.Particularly useful are carriers capable of forming a film or layer overthe skin to localize application and inhibit removal. For topicaladministration to internal tissue surfaces, the α-Gal A preparation maybe dispersed in a liquid tissue adhesive or other substance known toenhance adsorption to a tissue surface. For example, several mucosa)adhesives and buccal tablets have been described for transmucosal drugdelivery, such as in U.S. Pat. Nos. 4,740,365, 4,764,378, and 5,780,045,each incorporated herein by reference.

Hydroxypropylcellulose or fibrinogen/thrombin solutions may also beincorporated. Alternatively, tissue-coating solutions, such aspectin-containing formulations may be used. The preparations of theinvention may be provided in containers suitable for maintainingsterility, protecting the activity of the active ingredients duringproper distribution and storage, and providing convenient and effectiveaccessibility of the preparation for administration to a patient. Aninjectable formulation of an α-Gal A preparation might be supplied in astoppered vial suitable for withdrawal of the contents using a needleand syringe. The vial would be intended for either single use ormultiple uses. The preparation can also be supplied as a prefilledsyringe. In some instances, the contents would be supplied in liquidformulation, while in others they would be supplied in a dry orlyophilized state, which in some instances would require reconstitutionwith a standard or a supplied diluent to a liquid state. Where thepreparation is supplied as a liquid for intravenous administration, itmight be provided in a sterile bag or container suitable for connectionto an intravenous administration line or catheter. In preferredembodiments, the preparations of the invention are supplied in eitherliquid or powdered formulations in devices which conveniently administera predetermined dose of the preparation; examples of such devicesinclude a needle-less injector for either subcutaneous or intramuscularinjection, and a metered aerosol delivery device. In other instances,the preparation may be supplied in a form suitable for sustainedrelease, such as in a patch or dressing to be applied to the skin fortransdermal administration, or via erodible devices for transmucosaladministration. In instances where the preparation is orallyadministered in tablet or pill form, the preparation might be suppliedin a bottle with a removable cover. The containers may be labeled withinformation such as the type of preparation, the name of themanufacturer or distributor, the indication, the suggested dosage,instructions for proper storage, or instructions for administration.

Methods of Administration of α-Gal A Preparation

The α-Gal A preparations described herein may be administered by anyroute which is compatible with the α-Gal A preparation. The purifiedα-Gal A preparation can be administered to individuals who produceinsufficient or defective α-Gal A Protein or who may benefit from α-GalA therapy. Therapeutic preparations of the present invention may beprovided to an individual by any suitable means, directly (e.g.,locally, as by injection, implantation or topical administration to atissue locus) or systemically (e.g., orally or parenterally).

The preferred route of administration is intravenous. Other routes ofadministration may be oral or parenteral, including subcutaneous,intra-arterial, intraperitoneal, ophthalmic, intramuscular, buccal,rectal, vaginal, intraorbital, intracerebral, intradermal, intracranial,intraspinal, intraventricular, intrathecal, intracistemal,intracapsular, intrapulmonary, intranasal, transmucosal, transdermal, orvia inhalation. Intrapulmonary delivery methods, apparatus and drugpreparation are described, for example, in U.S. Pat. Nos. 5,785,049,5,780,019, and 5,775,320, each incorporated herein by reference. Apreferred method of intradermal delivery is by iontophoretic deliveryvia patches; one example of such delivery is taught in U.S. Pat. No.5,843,015, which is incorporated herein by reference.

A particularly useful route of administration is by subcutaneousinjection. An α-Gal A preparation of the present invention is formulatedsuch that the total required dose may be administered in a singleinjection of one or two milliliters. In order to allow an injectionvolume of one or two milliliters, an α-Gal A preparation of the presentinvention may be formulated at a concentration in which the preferreddose is delivered in a volume of one to two milliliters, or the α-Gal Apreparation may be formulated in a lyophilized form, which isreconstituted in water or an appropriate physiologically compatiblebuffer prior to administration. Subcutaneous injections of α-Gal Apreparations have the advantages of being convenient for the patient, inparticular by allowing self-administration, while also resulting in aprolonged plasma half-life as compared to, for example, intravenousadministration. A prolongation in plasma half-life results inmaintenance of effective plasma α-Gal A levels over longer time periods,the benefit of which is to increase the exposure of clinically affectedtissues to the injected α-Gal A and, as a result, may increase theuptake of α-Gal A into such tissues. This allows a more beneficialeffect to the patient and/or a reduction in the frequency ofadministration. Furthermore, a variety of devices designed for patientconvenience, such as refillable injection pens and needle-less injectiondevices, may be used with the α-Gal A preparations of the presentinvention as discussed herein.

Administration may be by periodic injections of a bolus of thepreparation, or may be administered by intravenous or intraperitonealadministration from a reservoir which is external (e.g., an IV bag) orinternal (e.g., a bioerodable implant, a bioartificial organ, or apopulation of implanted α-Gal A production cells). See, e.g., U.S. Pat.Nos. 4,407,957 and 5,798,113, each incorporated herein by reference.Intrapulmonary delivery methods and apparatus are described, forexample, in U.S. Pat. Nos. 5,654,007, 5,780,014, and 5,814,607, eachincorporated herein by reference. Other useful parenteral deliverysystems include ethylene-vinyl acetate copolymer particles, osmoticpumps, implantable infusion systems, pump delivery, encapsulated celldelivery, liposomal delivery, needle-delivered injection, needle-lessinjection, nebulizer, aeorosolizer, electroporation, and transdermalpatch, needle-less injector devices are described in U.S. Pat. Nos.5,879,327; 5,520,639; 5,846,233 and 5,704,911, the specifications ofwhich are herein incorporated by reference. Any of the α-Gal Apreparation described above can administered in these methods.

The route of administration and the amount of protein delivered can bedetermined by factors that are well within the ability of skilledartisans to assess. Furthermore, skilled artisans are aware that theroute of administration and dosage of a therapeutic protein may bevaried for a given patient until a therapeutic dosage level is obtained.

All patents and publications cited in this specification areincorporated by reference.

EXAMPLES Example 1 Preparation and Use of Constructs Designed to Deliverand Express α-Gal A

1.1: Preparation of Gene-Activated α-Gal A (GA-GAL)

Production of gene-activated α-Gal A (GA-GAL) occurred by insertion ofregulatory and structural DNA sequences upstream of the human α-Gal Acoding sequence, using the GA technology substantially as described inU.S. Pat. No. 5,733,761, herein incorporated by reference. The preciseinsertion of the gene-activating sequence occurs as a result ofhomologous recombination between DNA present on a transfected DNAfragment and genomic DNA sequences upstream of the α-Gal A locus in ahuman cell. The gene-activating sequence itself contains α-Gal A codingsequence up to, but not including, the signal peptide cleavage site.Cells containing an activated α-Gal A locus were isolated and subjectedto drug selection to isolate cells with increased GA-GAL production.

A targeting DNA fragment containing an appropriate gene-activatingsequence was introduced into host human cell lines by electroporation.One such cell line is HT-1080, a certified cell line available from ATCC(Manassas, Va.). The gene activation plasmid (targeting construct)pGA213C containing such a DNA fragment is shown in FIG. 3A. This plasmidcontains sequences designed to activate a portion of the endogenousα-Gal A locus in the host cell line, and contains sequences encoding thesignal peptide, but not human α-Gal A. The targeting construct alsocontains expression cassettes for the bacterial neo and mouse αGal Agenes. These allow for the selection of stably integrated targetingfragments (via the neo gene) and for subsequent selection of the dhfrgene using step-wise methotrexate (MTX) selection.

In addition, pGA213C contains sequences designed to target chromosomalsequences upstream of the endogenous α-Gal A locus by homologousrecombination. Homologous recombination between the endogenous α-Gal Alocus and the 9.6 kb DNA fragment of pGA213C is shown in FIG. 3B.

pGA213C was constructed to delete 962 bp of genomic sequences extendingfrom positions −1183 to −222 relative to the methionine initiation codonof α-Gal A, upon homologous recombination of the pGA213C fragment withthe X-chromosomal α-Gal A locus. Transcriptional activation of theα-Gal. A locus occurs through precise targeting of the exogenousregulatory sequences upstream of the α-Gal A coding region. Theresulting GA-GAL locus cause transcription to initiate from the CMVpromoter and to proceed through CMV exon 1, the aldolase intron and theseven exons and six introns of the α-Gal A coding sequence. Splicing ofthe large precursor mRNA joins the exogenous CMV exon (inserted bytargeting) with the entire endogenous first exon of α-Gal A transcript.Translation of the GA-GAL mRNA results in pre. GA-GAL with a thirty oneamino acid signal peptide. Upon secretion from the host cell, the signalpeptide is removed. Correctly targeted cell lines are first identifiedby polymerase chain reaction screening for the presence of the GA-GALmRNA. Clones producing the GA-GAL mRNA are also found to secreteenzymatically active α-Gal A into the culture media. Subsequentconfirmation of targeting events is accomplished by restriction enzymedigestion and Southern blot hybridization analysis of genomic DNA.

Cells were exposed to stepwise methotrexate (“MTX”) selection. Followingselection in 0.05 μM MTX, a clone of cells was isolated and subjected to0.1 μM MTX selection. From this process a pool of cells resistant to 0.1μM MTX was isolated (cell line RAG001) and expanded in culture.

1.2: Preparation of Other Constructs to Express α-Gal A

Two other expression plasmids, pXAG-16 and pXAG-28, were constructed.These plasmids contain human α-Gal A cDNA encoding the 398 amino acidsof the α-Gal A enzyme (without the α-Gal A signal peptide); the humangrowth hon-none (hGH) signal peptide genomic DNA sequence, which isinterrupted by the first intron of the hGH gene; and the untranslatedsequence (UTS) of the hGH gene, which contains a signal forpolyadenylation. Plasmid pXAG-16 has the human cytomegalovirusimmediate-early (CMV IE) promoter and first intron (flanked bynon-coding exon sequences), while pXAG-28 is driven by the collagen Iα2promoter and exon 1, and also contains the β-actin gene's 5′UTS, whichcontains the first intron of the β-actin gene.

In order to express α-Gal A in fibroblasts, secondary fibroblasts werecultured and transfected according to published procedures (Selden etal., WO 93/09222). The plasmids pXAG-13, pXAG-16 and pXAG-28 weretransfected by electroporation into human foreskin fibroblasts togenerate stably transfected clonal cell strains, and the resulting α-GalA expression levels were monitored. Secretion of α-Gal A by normalforeskin fibroblasts is in the range of 2-10 units/10⁶ cells/24 hours.In contrast, the transfected fibroblasts displayed mean expressionlevels as shown in the table below.

Mean α-Gal A expression levels (±standard deviations) pXAG-13: 420 ± 344U/10 ⁶ cells/day N = 26 clonal strains (range 3-1133 U/10 ⁶ cells/day)pXAG-16: 2,051 ± 1253 U/10 ⁶ cells/day N = 24 clonal strains (range422-5200 U/10 ⁶ cells/day) pXAG-28: 141 ± 131 U/10 ⁶ cells/day N = 38clonal strains (range 20-616 U/10 6 cells/day)

These data show that all three expression constructs are capable ofincreasing α-Gal A expression many times that of nontransfectedfibroblasts. Expression by fibroblasts stably transfected with pXAG-13,which encodes α-Gal A linked to the α-Gal A signal peptide, wassubstantially lower than expression by fibroblasts transfected withpXAG-16, which differs only in that the signal peptide is the hGH signalpeptide, the coding sequence of which is interrupted by the first intronof the hGH gene.

Cell strains desirable for gene therapy or for use in generation ofmaterial for purification of α-Gal A should display stable growth andexpression over several passages. Data from the cell strains which werestably transfected with the α-Gal A expression construct showed thatα-Gal A expression is stably maintained during serial passage.

Example 2 Structural Comparison of α-Gal A Produced in Human Cells vs.CHO Cells

This example compares the structure of Replagal™, an α-Gal A preparationproduced in human cells vs. Fabrazyme™, an α-Gal A preparation producedin CHO cells. The preparations were compared with respect to isoelectricpoint, molecular weight, and carbohydrate, phosphorylation andsialylation profile.

Isolelectric Point

Replagal™ and Fabrazyme™ were analyzed by denaturing isoelectricfocusing (5% gels, pH range 3 to 7, 6 M urea) followed by Westernblotting. The preparations were also analyzed by native isoelectricfocusing (Novex 5% gels, pH range 3 to 7) following by Coomassie Bluestaining. The overall pI range of the 2 preparations were similar,although the relative intensities of the banding patterns weredifferent. This indicates that the glycoforms present in eachpreparation differ in charge distribution, with Fabrazyme™ containing agreater proportion of lower pI (more negatively charged) glycoforms thanReplagal™.

Molecular Weight

Replagal™ and Fabrazyme™ were analyzed by SDS-PAGE (8-16% polyacrylamidegel, reduced samples) followed by Coomassie Blue staining. The molecularweights of the preparations were similar. However, the lower(approximately 45 kD) glycoform band of Fabrazyme™ is more distinctcompared to that of Replagal™, while Replagal™ exhibits a broader sizedistribution.

In FIG. 6, The molecular masses of Replagal™ (top) and Fabrazyme™(bottom) were determined by MALDI-TOF mass spectroscopy. The maximum ofthe major broad peak is at 50,755 and 50,705 Da, respectively,consistent with the expected molecular weight of the glycosylatedmonomer. A leading shoulder at approximately 48,071 Da and 47,667 Da ispresent, representing the lower molecular weight glycoforms forReplagal™ and Fabrazyme™, respectively. The leading shoulder,corresponding to the lower molecular weight glycoforms, is much moredistinct in the spectrum of Fabrazyme™.

FIG. 8 shows Fabrazyme™ (top) and Replagal™ (bottom) as analyzed byreversed phase HPLC using a C4 reversed phase column (Vydac).Chromatograms obtained at 214 nm are shown. The leading shoulder,corresponding to the lower molecular weight glycoforms, is much morepronounced in Fabrazyme™.

Cellular Internalization

Normal human fibroblasts were incubated in multi-well culture plates for6 hours in the absence (control, not shown) or presence of Replagal™ orFabrazyme™. This internalization is mannose-6-phosphate inhibitable,indicating that internalization is predominantly via mannose-6-phosphatereceptors. The results indicate that Replagal™ and Fabrazyme™ are notinternalized comparably by the fibroblasts. Fabrazyme™ is internalizedmore rapidly than Replagal™ by mannose-6-phosphate receptor-mediatedinternalization (see FIG. 5).

Glycan Composition and Characterization

FIG. 7 shows charge profiles of the glycans released from Replagal™(bottom) and Fabrazyme™ (top). Glycans were derivatized with afluorescent probe and compared by ion exchange chromatography on aGlycoSep™ C column. The results show that Replagal™ has a higherproportion of neutral and mono-charged glycans, and Fabrazyme™ has ahigher proportion of tri-charged glycans.

Table 1 shows a glycan peak area comparison. Glycans released fromReplagal™ and Fabrazyme™ were analyzed using HPAE-PAD as shown in FIG.4. Integration of peaks was performed to quantify the percentages of thevarious peak groups. The tabulated data demonstrate a higher proportionof phosphorylated glycans in Fabrazyme™, and a higher proportion ofneutral glycans and a higher total proportion of sialylated glycans inReplagal™.

Table 2 shows charge profile results. Charge profiles of glycansreleased from Replagal™ and Fabrazyme™ were derivatized and separated asdescribed in FIG. 7. CK-022 and CK-006 are 2 different Replagal™preparations, while CK-JL012502 is a preparation of Fabrazyme™. Glycansfrom each product were assayed in duplicate. As shown in the table, theReplagal™ preparations have higher proportions of glycans that areneutral or carry 1 charge, while Fabrazyme™ contains a higher proportionglycans with 2 or 3 charges.

Table 3 shows the desialylated profile results. Charge profiles ofglycans released from Replagal™ and Fabrazyme™ were desialylated,followed by derivatization and separation as described in FIG. 7. CK-022and CK-006 are 2 different Replagal™ preparations, while CK-JL012502 isa preparation of Fabrazyme™. Glycans from each product were assayed induplicate. As shown in the table, the Replagal™ preparations have lowerproportions of residual charged glycans after desialylation, indicatingthat Fabrazyme™ has a higher proportion of phosphorylated(sialidase-resistant) glycans.

Example 3 Interspecies Scaling of Pharmacokinetics of α-Gal A(Replagal™) Made in Human Cells

The purpose of this example was to compare pharmacokinetic parametersderived from animal models with human pharmacokinetic results.

Animals (mice, rats, dogs, rabbits and monkeys) received singleintravenous bolus injections of Replagal™. Blood samples were collectedover a 24 hour period, processed to serum, and analyzed for α-Gal Aenzyme activity using an in vitro fluorescence assay. Serumconcentration profiles were analyzed using either a 2-compartment modelor a noncompartmental model to estimate pharmacokinetic parameters.

Blood samples were collected from male Fabry patients receiving theirinitial 40 minute infusion of Replagal™. Blood samples were processed toeither plasma or serum and analyzed for α-Gal A enzyme activity. Serumconcentration profiles were analyzed using a noncompartment model toestimate pharmacokinetic parameters.

Liver biopsies were taken 44 hours after dosing from male Fabry patientsin the Phase I trial. Tissue samples were processed and analyzed forconcentration of administered α-Gal A as previously described (Schiffmanand Brady et al. (2000) Proc. Natl. Acad. Sci. USA 97:365-370). Theamount of administered dose recovered in each patient's liver wascalculated using the concentration of α-Gal A in each liver biopsy andeach patient's estimated liver weight.

Replagal™ had a biphasic serum elimination profile following a single IVdose in rats, rabbits and monkeys (FIG. 9 illustrates the profile incynomolgus monkey). C_(max) was proportional to dose for these threeanimal species (FIG. 10). Replagal™ also had a biphasic serumelimination profile in Fabry patients following a 40 minute infusion(FIG. 11). C_(max) was also dose proportional in humans (FIG. 12).Replagal™ was eliminated by 24 hours after dosing in all species. AUC(area under the curve) increased linearly with dose in animals andhumans over a dose range of 0.017 to 3.2×10⁶ U/kg (FIG. 13). The doserange in U/kg corresponds to a range of 0.007 to 0.2 mg/kg in humans and0.0625 to 1 mg/kg in animals.

Physiological parameters in mammals follow allometric scaling equationsbased on body weight, Y=a (BW)^(b) (Table 4). The exponent in thescaling equation can be near 1.0 (e.g., blood volume) but varies between0.6 and 0.8 for drug or protein clearance.

The allometric scaling equation of Replagal™ (mL/min) was based onpharmacokinetic studies in mice, rats, rabbits, large and smallcynomolgus monkeys, and Fabry patients. Serum clearance followed theallometric scaling equation with an exponent of 0.92. (Table 5). Theincreased exponent for Replagal™ serum clearance in comparison to otherdrug products or proteins provides support for M6P receptor clearance ofReplagal™.

The percent of administered Replagal™ found in a patient's liverdecreased as the dose increased on a mg/kg basis (Table 6). At the twolowest doses, 0.007 and 0.014 mg/kg, the percent of Replagal™ recoveredin the liver 44 hours after dosing was approximately 25 to 30%. Incontrast, at 0.11 mg/kg, only 14% of administered Replagal™ was found inthe liver. Saturation of liver uptake of Replagal™ occurred when maximumdrug product concentrations (C_(max)) exceeded the Kd for the M6Preceptor (2×10⁻⁹ M). Based on these results, the estimated amount of thecommercial dose of Replagal™ (0.2 mg/kg) taken into liver isapproximately 2 mg for a 75 mg patient (FIG. 14). the remainder of thedose (13 mg) would then be available for uptake into tissues other thanliver.

Thus, single dose pharmacokinetics in animal models provided a goodprediction of Replagal™ pharmacokinetics in Fabry patients. Themechanism of clearance of Replagal™ from blood is predominantly throughM6P receptors which are found in tissues throughout the body. Theexponent for the allometric scaling equation for serum or plasmaclearance of Replagal™, 0.92, is greater than that observed for otherdrug products or proteins. The increased exponent provides support forM6P receptor clearance of Replagal™. Saturation of human liver receptorswas observed when C_(max) exceeded the Kd of the M6P receptor (doses of0.056 mg/kg and higher).

Example 4 Pharmacokinetics of Replagal™ in Male and Female FabryPatients

The primary purpose of this evaluation was to compare Replagal™pharmacokinetic properties in male and female Fabry patients. Asecondary objective was to compare pharmacokinetic properties betweenpatients treated with Replagal™ and Fabrazyme™.

Blood samples were collected from male and female Fabry patientsreceiving their initial 40 minute infusion of Replagal from TKT006(NIH), TKT007 (UK) and TKT014 (GERMANY). Blood samples were processed toserum (TKT007 and TKT014 samples) or plasma (TKT006 samples) andanalyzed for α-galactosidase A enzyme activity at TKT using an in vitrofluorescence assay. Serum/plasma concentration profiles were analyzedusing a noncompartmental model to estimate pharmacokinetic parameters.

Predose enzyme activity averaged 1.2 U/ml in males and 6.5 U/ml infemales which reflects the carrier status of female patients (Table 7).

Replagal™ had a biphasic serum elimination profile following a singleintravenous infusion in both male and female Fabry patients and waseliminated from most patients by 24 hours after dosing (FIG. 15). Asexpected, C_(max) coincided with the end of the 40 minute infusionperiod.

Mean pharmacokinetic parameters were similar between male and femalepatients (Table 8). AUC (area under the curve) normalized for dose wasslightly greater in females (ratio of 0.51) but was not statisticallydifferent from the male ration (0.43). Absolute serum clearance ofReplagal™ was lower in females (140 compared to 177 mL/min); but whennormalized for body weight, serum clearance was not statisticallydifferent (2.10 versus 2.52 mL/min/kg). The statistically significantdifference in terminal elimination half-life (89 minutes in femalesversus 112 minutes in males) was not due to a difference in eliminationof Replagal™ from females. Instead, the higher baseline enzyme activityin women made it difficult to detect administered Replagal™ beyond 8hours (T_(last)).

Clearance of Replagal™ from the circulation of Fabry patients was morerapid than individual patient GFR or creatinine clearance which isconsistent with its mechanism of clearance (Table 9). Replagal™ isprimarily cleared from the circulation by uptake into tissues viamannose-6-phosphate (M6P) receptors and minimally by protein degradationand kidney elimination.

An analysis was performed to confirm that changes in renal function willnot affect clearance of Replagal™ from the circulation (Table 10). Mostof the Fabry patients undergoing first dose pharmacokinetic analysiswere either in the normal range (>80 mL/min creatine clearance) or had“mild” renal impairment (50-80 mL/min creatine clearance) when theyreceived their first dose of Replagal™. Although only 5 patients were inthe moderate or severe categories, serum clearance of Replagal(ml/min/kg) for these patients was within the range established by the 2higher renal function categories. These data suggest that Replagal™ isnot excreted by the kidney. There were no differences between males andfemales in this analysis.

Serum clearance of Fabrazyme™ from Fabry patients was significantly morerapid compared to that observed with Replagal™ (Table 11). At nearlyequal doses in male patients (0.2 and 0.3 mg/kg for Replagal andFabrazyme, respectively), serum clearance of Fabrazyme™ was 4 mL/min/kgcompared to 2.5 mL/min/kg for Replagal™. This difference in serumclearance at nearly equivalent doses is due to the differentglycosylation pattern of Fabrazyme™ (manufactured in CHO cells) comparedto the human glycosylation pattern of Replagal™. At higher doses ofFabrazyme™ (1 and 3 mg/kg), serum clearance was significantly reduced toapproximately 2.7 and 1 mL/min/kg as clearance mechanisms becamesaturated for Fabrazyme™.

Thus, pharmacokinetic parameters were similar in male and femalepatients dosed with Replagal™. Serum clearance of Replagal™significantly exceeded renal function (mL/min), consistent with M6Pmediated uptake of Replagal™ into tissues and cells throughout the body.As expected, preliminary analysis indicated that Replagal is notexcreted by the kidney. At doses below clearance saturation levels,Fabrazyme™ serum clearance was significantly more rapid compared toReplagal™ and reflects differences in glycosylation patterns between thetwo drug products.

TABLE 1 Replagal Fabrazyme Neutral 15.6%  4.6% 1 Sialic Acid 29.5%  8.9%Unknown  4.6%  1.8% 2 Sialic Acid 20.1% 11.3% 1 Phosphate 13.0% 27.1%Unknown  4.0%  4.6% 3 Sialic Acid  1.9%  8.5% 4 Sialic Acid  5.9% 15.1%2 Phosphate  5.4% 18.0%

TABLE 2 % % 1 % 2 % 3 % 4 Sample ID Neutrals Charges Charges ChargesCharges CK-022 vial 1 32.76 26.97 25.64 9.78 4.85 CK-022 vial 2 33.6126.37 25.16 9.92 4.93 CK-006 vial 1 29.73 25.61 27.12 11.85 5.70 CK-006vial 2 29.33 26.69 27.14 11.47 5.37 CK-JL012502 21.88 12.59 38.44 21.945.15 vial 1 CK-JL012502 21.60 12.61 39.35 21.44 5.00 vial 2

TABLE 3 Sample ID % Neutrals % 1 Charges % 2 Charges CK-022 inj 1 94.452.73 2.87 CK-022 inj 2 94.39 2.68 2.93 CK-006 inj 1 93.18 2.57 4.25CK-006 inj 2 93.34 2.24 4.42 CK-JL012502 inj 1 88.52 4.37 7.11CK-JL012502 inj 2 88.08 4.41 7.51

TABLE 4 Allometri Scaling of Physiological and Anatomical Parameters(function of body weight, BW) Equation: Y = a (BW) ^(b) Parameter (Y)Exponent (b) Body surface area 0.67 Blood volume (mL) 0.99 Lung weight(g) 0.99 Urine output (mL/h) 0.82 Insulin clearance (mL/h) 0.77 Kidneyweight (g) 0.85 Drug Clearance from Plasma 0.6-0.8 Protein Clearancefrom Plasma 0.65-0.84 M6P Receptor Clearance ? Chappell and Mordenti(1991) Extrapolation of Toxicological and Pharmacological Data fromAnimals to Humans. In B. Testa (ed.) Advances in Drug Research, Vol. 20,pp. 1-116.

TABLE 5 Allometric Scaling of Pharmacokinetic Parameters-Exponents DrugProduct Cl (mL/min) Small molecules* 0.6-0.8 Published proteins*0.65-0.84 Rt-PA 0.84 Relaxin 0.80 CD4-IgG 0.74 rhGH 0.71 RCD4 0.65Replagal   ^(TM) 0.92 *Mordenti et al. (1991) Interspecies Scaling ofClearance and Volume Distribution Data for Five Therapeutic Proteins.Pharmaceutical Research 8: 1351-1359.

TABLE 6 Percent Administered Dose of Replagal in Human Liver Percentadministered Dose in Liver T _(max) (2 hours) Dose Infusion C _(max) 44Hr claculated using (mg/kg) Time (×10 ⁻ ⁹ M) (measured) T _(1/2) or 3Days 0.007 20 min 0.4 25.0% 38% 0.014 20 min 1.1 28.7% 43% 0.028 20 min2.5 16.8% 25% 0.056 20 min 5.0 19.4% 29% 0.11 20 min 7.8 13.7% 21% 0.240 min 11.6 not determined 8%-18% (estimate) M6P receptor Kd is 2 × 10 ⁻⁹ M (Komfeld Ann Rev Biochem 61:307-330, 1992) α-Galactosidase Ahalf-lives: 4 days in Fabry fibroblasts (Mayes et al., Am J Hun, Genet34:602-610, 1982) 2 days in mouse liver (Ioannou et al:, Am J Hum Genet68:14-25, 2001)

TABLE 7 Baseline Values of α-Galactosidase A Enzyme Activity AverageBaseline Value Study No. No. of Patients (U/mL) TKT006 and TKT007 39males 1.2* (range 0.4-9) (NIH and UK) TKT014 (Germany) 15 females 6.5(range 2-12) *excludes one male patient with a baseline value ofapproximately 15 U/mL 1 Unit (U) is defined as the hydrolysis of onenanomole of 4-methylumbelliferyl-α-D-galactopyranoside per hour at 37°C.

TABLE 8 Pharmacokinetic Comparison between Male and Female FabryPatients Following 1^(st) Dose of Replagal Avg Body Clinical Dose Wt CICI T_(1/2) (λ_(z)) Median V_(ss) Study No. (U/kg × 10⁶) (kg) AUC/Dose(mL/min) (mL/min/kg) (min) T_(last) (% BW) TKT006 18 0.61 72.9 0.43 177 2.52 112  12 16.0% TKT007 males (16.1) (0.12) (43) (0.74) (25) hours (4.3%) TKT014 15 0.66 68.0 0.51 140  2.10 89  8 16.5% females (13.8)(0.13) (38) (0.62) (28) hours  (4.3%) t-test  0.057    0.015 0.10   0.02 NA NS ( ) standard deviation NA, not applicable NS, notsignificant T_(last), time of last detectable Replagal enzyme activityNormalized AUC has units of (min*U/mL)/(U/kg)

TABLE 9 Serum Clearance of Replagal from Male and Female Patients MeanReplagal Pharmacokinetic Mean GFR* Clearance Study No. Evaluation (N)(mL/min) (mL/min) TKT006 (NIH) 10 males 78 (24) 193 (47) TKT005 (UK)  8males 115 (63) 157 (29) Combined 18 males 95 (48) 177 (43) TKT014(Germany) 15 females 70 (20)† 140 (38) ( ) standard deviation N, numberof patients evaluated for pharmacokinetic parameters following firstdose of Replagal *GFR, glomerular filtration rate, measured 2-3 weeksbefore first dose of Replagal †creatinine clearance measured in female

TABLE 10 Comparison of Renal Function and Replagal Serum Clearance Rangeof Replagal Pharmacokinetic Real Function Clearance Study No. Evaluation(N) Category* (mL/min/kg) TKT006 (NIH) 10 males Normal Range 1.7-3.4 andTKT007 (UK) (>80mL/min)  6 males Mild 2.0-4.4 (50-80 mL/min)  1 malesModerate 3.5 (30-50 mL/min)  1 males Severe 1.6 (<30 mL/min) TKT014 5females Normal Range 2.2-3.0 (Germany) (>80 mL/min) 7 females Mild1.4-3.6 (50-80 mL/min) 2 females Moderate 1.5-1.6 (30-50 mL/min) 1females Severe 1.8 (<30 mL/min) *FDA categories based on estimatedcreatinine clearance

TABLE 11 Serum Clearance in Fabry Patients Dosed with Replagal orFabrazyme Serum Clearance (mL/min/kg) Replagal Dose (mg/kg) FabrazymeMales* Males Females 0.2 2.5 2.1 0.3 4 1.0 ~2.7† 3.0   ~1† *Eng et al(2001) A Phase I/II Clinical Trial of Enzyme Replacement in FabryDisease. Am J Hum Genet 68:711-722. †serum clearance saturated

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references disclosed herein are incorporated by reference in theirentirety. What is claimed is presented below and is followed by aSequence Listing.

We claim:
 1. A method of producing a pharmaceutical compositioncomprising human α-Gal A, the method comprising the steps of: obtaininga first batch of a human α-Gal A preparation harvested from a cell;obtaining a sample from the batch; determining if the sample has four ormore of the following characteristics: (1) has at least about 75%neutral, mono- and di-sialylated glycans combined; (2) has less thanabout 35% tri- and tetra-sialylated glycans combined; (3) has greaterthan 50% complex glycans; (4) has less than about 45% phosphorylatedglycans; (5) has greater than about 45% sialylated glycans; (6) has aratio of sialic acid to mannose-6-phosphate on a mole per mole basisgreater than 1.5 to 1; and (7) has a ratio of sialylated glycans tophosphorylated glycans greater than 1, selecting the batch if the samplehas four or more of the characteristics (1)-(7); and formulating theselected batch in a pharmaceutically acceptable carrier, therebyproducing a pharmaceutical composition comprising human α-Gal A.
 2. Themethod of claim 1, wherein the determining step comprises determining ifthe sample has all of the characteristics (1)-(7) and the selecting stepcomprises selecting the batch if the sample has all of thecharacteristics (1)-(7).
 3. The method of claim 1, the determining stepfurther comprising entering the result of the determination into arecord.
 4. The method of claim 1, wherein the determining step isperformed by one or more methods selected from the group consisting ofion exchange chromatography, high performance anion exchange (HPAE)chromatography, high performance liquid chromatography (HPLC), and massspectroscopy.
 5. The method of claim 1, wherein the batch is harvestedfrom a mammalian cell.
 6. The method of claim 5, wherein the mammaliancell is a human cell.
 7. The method of claim 5, wherein the mammaliancell is a non-human cell.
 8. The method of claim 5, wherein themammalian cell is a CHO cell.
 9. The method of claim 1, the methodfurther comprising the steps of: obtaining a second batch of a humanα-Gal A preparation; obtaining a sample from the second batch;determining if the sample from the second batch has four or more of thecharacteristics (1)-(7); and formulating the second batch in apharmaceutically acceptable carrier.
 10. The method of claim 9, whereinthe first and second batches are first and second batches of the samehuman α-Gal A preparation.
 11. The method of claim 9, the method furthercomprising the step of selecting the second batch having four or more ofthe characteristics (1)-(7) prior to formulating said second batch. 12.The method of claim 1, the method further comprising the step ofmodifying the glycan structure of the first batch to match four or moreof the characteristics prior to formulating said first batch.
 13. Themethod of claim 1, the method further comprising the step of purifyingthe first batch having four or more of the characteristics (1)-(7) priorto formulating said first batch.
 14. The method of claim 1, wherein theobtaining step comprises isolating a human α-Gal A preparation from acell or cell culture.
 15. The method of claim 14, wherein the obtainingstep further comprises lysing the cell.
 16. The method of claim 14,wherein the obtaining step further comprises collecting the culturemedium in which the cell is grown.
 17. The method of claim 14, whereinthe cell is a human cell genetically engineered to produce human α-GalA.
 18. The method of claim 1, the obtaining step further comprising thestep of purifying the α-Gal A preparation.
 19. The method of claim 1,the formulating step comprising formulating the batch in a buffercomprising phosphate, sodium chloride, and polysorbate-20.
 20. Themethod of claim 1, the method further comprising the step oflyophilizing the formulated batch.
 21. A method of producing apharmaceutical composition comprising human α-Gal A, the methodcomprising the steps of: isolating a human α-Gal A preparation from acell or cell culture; purifying the α-Gal A preparation; obtaining asample from the α-Gal A preparation; determining if the sample has fouror more of the following characteristics: (1) has at least about 75%neutral, mono- and di-sialylated glycans combined; (2) has less thanabout 35% tri- and tetra-sialylated glycans combined; (3) has greaterthan 50% complex glycans; (4) has less than about 45% phosphorylatedglycans; (5) has greater than about 45% sialylated glycans; (6) has aratio of sialic acid to mannose-6-phosphate on a mole per mole basisgreater than 1.5 to 1; and (7) has a ratio of sialylated glycans tophosphorylated glycans greater than 1, selecting the α-Gal A preparationif the sample has four or more of the characteristics (1)-(7); andformulating the selected α-Gal A preparation in a pharmaceuticallyacceptable carrier, thereby producing a pharmaceutical compositioncomprising human α-Gal A.
 22. The method of claim 21, the method furthercomprising the step of culturing a cell genetically engineered toproduce human α-Gal A prior to the isolating step.
 23. The method ofclaim 21, further comprising lyophilizing the formulated α-Gal Apreparation.